Intron double stranded RNA constructs and uses thereof

ABSTRACT

The present invention is in the field of plant genetics and provides agents capable of gene-specific silencing. The present invention specifically provides double-stranded RNA (dsRNA) agents, methods for utilizing such agents and plants containing such agents.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit under 35 U.S.C. § 119(e) ofU.S. Provisional Application No. 60/390,186, filed Jun. 21, 2002, whichapplication is herein incorporated by reference in its entirety.

INCORPORATION OF SEQUENCE LISTING

[0002] A paper copy of the Sequence Listing and a computer readable formof the sequence listing on diskette, containing the file named “RNAi16517266 US as filed.txt”, which is 60,564 bytes in size (measured inMS-DOS), and which was created on Jun. 19, 2003, are herein incorporatedby reference.

FIELD OF THE INVENTION

[0003] The present invention is in the field of plant genetics andprovides agents capable of gene-specific silencing. The presentinvention specifically provides double stranded RNA (dsRNA) agents,methods for utilizing such agents and plants containing such agents.

BACKGROUND OF THE INVENTION

[0004] Silencing of genes in plants occurs at both the transcriptionallevel and post-transcriptional level. Certain of these mechanisms areassociated with nucleic acid homology at the DNA or RNA level (Matzke etal., Current Opinion in Genetics and Development, 11:221-227 (2001)).Double-stranded RNA molecules can induce sequence-specific silencing,referred to as RNA interference or RNAi. Fire et al., Nature,391:806-811 (1988).

SUMMARY OF THE INVENTION

[0005] The present invention includes and provides a nucleic acidconstruct comprising DNA which is transcribed into RNA that forms atleast one double-stranded RNA molecule, such that one strand of thedouble-stranded molecule is coded by a portion of the DNA which is atleast 90% identical to at least one transcribed intron of a gene.

[0006] The present invention also includes and provides a transformedcell or organism having in its genome an introduced nucleic acidconstruct comprising DNA which is transcribed into RNA that forms atleast one double-stranded RNA molecule, such that one strand of thedouble-stranded molecule is coded by a portion of the DNA which is atleast 90% identical to at least one transcribed intron of a gene.

[0007] The present invention further includes and provides a transformedplant having in its genome an introduced nucleic acid constructcomprising DNA which is transcribed into RNA that forms at least onedouble-stranded RNA molecule, such that one strand of thedouble-stranded molecule is coded by a portion of the DNA which is atleast 90% identical to at least one transcribed intron of a gene.

[0008] The present invention includes and provides a method of reducingexpression of a protein encoded by a target gene in a mammal comprisingintroducing into a cell or organism a nucleic acid construct comprisingDNA which is transcribed into RNA that forms at least onedouble-stranded RNA molecule, such that one strand of thedouble-stranded molecule is coded by a portion of the DNA which is atleast 90% identical to at least one transcribed intron of a gene.

[0009] The present invention includes and provides a method of reducingexpression of a protein encoded by a target gene in a plant comprisingintroducing into a plant genome a nucleic acid construct comprising DNAwhich is transcribed into RNA that forms at least one double-strandedRNA molecule, such that one strand of the double-stranded molecule iscoded by a portion of the DNA which is at least 90% identical to atleast one transcribed intron of a gene.

[0010] The present invention includes and provides a method of alteringthe expression of a target gene by inserting into a cell or organism aDNA construct for producing a double stranded RNA molecule coding for anintron within the target gene. More particularly, the nucleic acidconstruct comprises DNA which is transcribed into RNA that forms atleast one double-stranded RNA molecule, one strand of which is coded bya portion of DNA which is at least 90% identical to at least onetranscribed intron of a gene. In a preferred aspect of the invention,one strand of the double-stranded RNA molecule is at least 98%, evenmore preferably 100% identical, to an intron of a gene.

[0011] In one aspect of the invention, a construct for producingdouble-stranded RNA comprises one strand of an intron, a spliceableintron, and the complement of the intron, such that the spliceableintron provides a hairpin loop when the intron and the complement of theintron hybridize to each other.

[0012] In yet another aspect of this invention the constructs are basedon introns within a FAD2 gene or a FAD3 gene.

[0013] In yet another aspect of this invention the construct comprisesDNA which is transcribed into double-stranded RNA for at least twotranscribed introns, e.g. introns for two or three or more genes.

[0014] Another aspect of this invention provides a transformed cell ororganism having in its genome a nucleic acid construct which produces adouble-stranded RNA of a gene to be suppressed, e.g., in a plant or ananimal, preferably a plant, a mammal, an insect or a nematode. Thepresent invention provides a transformed plant having in its genome anucleic acid construct comprising DNA which is transcribed into RNA thatforms at least one double-stranded RNA molecule such that one strand ofthe double-stranded molecule is coded by a portion of the DNA which isat least 90% identical to at least one transcribed intron of a nativeplant gene or a plant pest gene.

[0015] This invention also provides a method of reducing expression of aprotein encoded by a target gene in a mammal comprising introducing intoa mammalian cell or organism a nucleic acid construct comprising DNAwhich produces double-stranded RNA based on an intron within a gene tobe suppressed. Another aspect of this invention provides a method ofreducing expression of a protein encoded by a target gene in a plantcomprising introducing into a plant cell or organism a nucleic acidconstruct comprising DNA which produces double-stranded RNA based on anintron within a gene to be suppressed.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016]FIG. 1 is a schematic of construct pCGN3892.

[0017]FIG. 2 is a schematic of construct pMON70674.

[0018]FIG. 3 is a schematic of construct pMON70678.

[0019]FIG. 4 is a schematic of construct pMON68546.

DETAILED DESCRIPTION OF THE INVENTION

[0020] Description of the Nucleic Acid Sequences

[0021] SEQ ID NO: 1 sets forth a nucleic acid sequence of a FAD2-1Aintron 1.

[0022] SEQ ID NO: 2 sets forth a nucleic acid sequence of a FAD2-1Bintron 1.

[0023] SEQ ID NO: 3 sets forth a nucleic acid sequence of a partialFAD2-2 genomic clone.

[0024] SEQ ID NO: 4 sets forth a nucleic acid sequence of a FAD2-2Bintron 1.

[0025] SEQ ID NO: 5 sets forth a nucleic acid sequence of a FAD3-1Aintron 1.

[0026] SEQ ID NO: 6 sets forth a nucleic acid sequence of a FAD3-1Aintron 2.

[0027] SEQ ID NO: 7 sets forth a nucleic acid sequence of a FAD3-1Aintron 3A.

[0028] SEQ ID NO: 8 sets forth a nucleic acid sequence of a FAD3-1Aintron 4.

[0029] SEQ ID NO: 9 sets forth a nucleic acid sequence of a FAD3-1Aintron 5.

[0030] SEQ ID NO: 10 sets forth a nucleic acid sequence of a FAD3-1Aintron 3B.

[0031] SEQ ID NO: 11 sets forth a nucleic acid sequence of a FAD3-1Aintron 3C.

[0032] SEQ ID NO: 12 sets forth a nucleic acid sequence of a FAD3-1Bintron 3C.

[0033] SEQ ID NO: 13 sets forth a nucleic acid sequence of a FAD3-1Bintron 4.

[0034] SEQ ID NO: 14 sets forth a nucleic acid sequence of a FAD3-1Cintron 4.

[0035] SEQ ID NO: 15 sets forth a nucleic acid sequence of a FAD2-1Agene sequence.

[0036] SEQ ID NOs: 16 and 17 set forth nucleic acid sequences of FAD2-1APCR primers.

[0037] SEQ ID NO: 18 sets forth a nucleic acid sequence of a partialFAD2-1A genomic clone.

[0038] SEQ ID NO: 19 sets forth a nucleic acid sequence of a partialFAD2-1B genomic clone.

[0039] SEQ ID NOs: 20 and 21 set forth nucleic acid sequences of FAD3-1APCR primers.

[0040] SEQ ID NO: 22 sets forth a nucleic acid sequence of a FAD2-1Bpromoter.

[0041] SEQ ID NO: 23 sets forth a nucleic acid sequence of a partialFAD3-1A genomic clone.

[0042] SEQ ID NOs: 24 through 39 set forth nucleic acid sequences of PCRprimers.

[0043] SEQ ID NO: 40 sets forth a nucleic acid sequence of a soybeanFATB genomic clone.

[0044] SEQ ID NO: 41 sets forth a nucleic acid sequence of a soybeanFATB intron I.

[0045] SEQ ID NO: 42 sets forth a nucleic acid sequence of a soybeanFATB intron II.

[0046] SEQ ID NO: 43 sets forth a nucleic acid sequence of a soybeanFATB intron III.

[0047] SEQ ID NO: 44 sets forth an amino acid sequence of a soybean FATBenzyme.

[0048] SEQ ID NO: 45 sets forth a nucleic acid sequence of a soybeanFATB partial genomic clone.

[0049] SEQ ID NOs: 46-53 set forth nucleic acid sequences ofoligonucleotide primers.

[0050] SEQ ID NO: 54 sets forth a nucleic acid sequence of a PCR productcontaining soybean FATB intron II.

[0051] SEQ ID NO: 55 sets forth a nucleic acid sequence of a soybeanFATB cDNA.

[0052] Definitions

[0053] As used herein, the term “gene” is used to refer to a nucleicacid sequence that encompasses a 5′ promoter region associated with theexpression of the gene product, any intron and exon regions and 3′untranslated regions associated with the expression of the gene product.

[0054] As used herein, a target gene can be any gene of interest presentin an organism which contains a transcribed intron. A target gene may beendogenous or introduced.

[0055] As used herein, when referring to proteins and nucleic acidsherein, the use of plain capitals, e.g., “FATB”, indicates a referenceto an enzyme, protein, polypeptide, or peptide, and the use ofitalicized capitals, e.g., “FA TB”, is used to refer to nucleic acids,including without limitation genes, cDNAs, and mRNAs.

[0056] As used herein, a cell or organism can have a family of more thanone gene encoding a particular enzyme. As used herein, a gene family istwo or more genes in an organism which encode proteins that exhibitsimilar functional attributes. An example of two members of a genefamily are FAD2-1 and FAD2-2. As used herein, a “FAD2 gene familymember” is any FAD2 gene found within the genetic material of the plant.As used herein, a “FAD3 gene family member” is any FAD3 gene foundwithin the genetic material of the plant. As used herein, a “FATB genefamily member” is any FATB found within the genetic material of theplant. A gene family can be additionally classified by the similarity ofthe nucleic acid sequences. In a preferred aspect of this embodiment, agene family member exhibits at least 60%, more preferably at least 70%,more preferably at least 80% nucleic acid sequence identity in thecoding sequence portion of the gene.

[0057] As used herein, RNAi and dsRNA both refer to gene-specificsilencing that is induced by the introduction of a double-stranded RNAmolecule, see e.g., U.S. Pat. Nos. 6,506,559 and 6,573,099, and U.S.patent applications 09/056,767 and 09/127,735, all of which areincorporated herein by reference.

[0058] As used herein, a “dsRNA molecule” and an “RNAi molecule” bothrefer to a double-stranded RNA molecule capable, when introduced into acell or organism, of at least partially reducing the level of an mRNAspecies present in a cell or a cell of an organism.

[0059] As used herein, an “intron dsRNA molecule” and an “intron RNAimolecule” both refer to a double-stranded RNA molecule capable, whenintroduced into a cell or organism, of at least partially reducing thelevel of an mRNA species present in a cell or a cell of an organismwhere the double-stranded RNA molecule exhibits sufficient identity toan intron of a gene present in the cell or organism to reduce the levelof an mRNA containing that intron sequence.

[0060] As used herein, a “FAD2”, “A 12 desaturase” or “omega-6desaturase” gene is a gene that encodes an enzyme capable of catalyzingthe insertion of a double bond into a fatty acyl moiety at the twelfthposition counted from the carboxyl terminus.

[0061] As used herein, the terminology “FAD2-1” is used to refer to aFAD2 gene that is naturally expressed in a specific manner in seedtissue.

[0062] As used herein, the terminology “FAD2-2” is used to refer a FAD2gene that is (a) a different gene from a FAD2-1 gene and (b) isnaturally expressed in multiple tissues, including the seed.

[0063] As used herein, a “FAD3”, “Δ15 desaturase” or “omega-3desaturase” gene is a gene that encodes an enzyme capable of catalyzingthe insertion of a double bond into a fatty acyl moiety at the fifteenthposition counted from the carboxyl terminus.

[0064] As used herein, the terminology “FAD3-1” is used to refer a FAD3gene that is naturally expressed in multiple tissues, including theseed.

[0065] As used herein, the capital letter that follows the geneterminology (A, B, C) is used to designate the family member, i.e.,FAD2-1A is a different gene family member from FAD2-1B.

[0066] The term “non-coding” refers to sequences of nucleic acidmolecules that do not encode part or all of an expressed protein.Non-coding sequences include but are not limited to introns, promoterregions, 3′ untranslated regions, and 5′ untranslated regions.

[0067] The term “intron” as used herein refers to the normal sense ofthe term as meaning a segment of nucleic acid molecules, usually DNA,that does not encode part of or all of an expressed protein, and which,in endogenous conditions, is transcribed into RNA molecules, but whichis spliced out of the endogenous RNA before the RNA is translated into aprotein. The splicing, i.e., intron removal, occurs at a defined splicesite, e.g., typically at least about 4 nucleotides, between cDNA andintron sequence. For example, without limitation, the sense andantisense intron segments illustrated herein, which form adouble-stranded RNA contained no splice sites.

[0068] The term “spliceable intron” as used herein refers to an intronthat contains functional splice sites at each end. For example, withoutlimitation, in the constructs illustrated herein, spliceable intronshave been used to form the hairpin loop connecting two antiparallel RNAstrands of intron sequence which had splice sites removed.

[0069] The term “exon” as used herein refers to the normal sense of theterm as meaning a segment of nucleic acid molecules, usually DNA, thatencodes part of or all of an expressed protein.

[0070] As used herein, a promoter that is “operably linked” to one ormore nucleic acid sequences is capable of driving expression of one ormore nucleic acid sequences, including multiple coding or non-codingnucleic acid sequences arranged in a polycistronic configuration.

[0071] As used herein, a “series” is a sequential collection of elementsarranged consecutively.

[0072] Nucleic Acid Molecules

[0073] Agents of the invention include nucleic acid molecules. In anaspect of the present invention, a nucleic acid molecule comprises anucleic acid sequence, which when introduced into a cell or organism, iscapable of selectively reducing the level of a target protein and/ortranscript that encodes a target protein.

[0074] In a preferred aspect, a nucleic acid molecule of the presentinvention exhibits sufficient homology to one or more introns which whenintroduced into a cell or organism as a dsRNA construct, is capable ofeffectively eliminating, substantially reducing, or at least partiallyreducing the level of an mRNA transcript or protein encoded by the genefrom which the intron was derived. In another preferred aspect, anucleic acid molecule of the present invention exhibits sufficienthomology to one or more introns such that, when introduced into a cellor organism as a dsRNA construct, the nucleic acid molecule is capableof effectively eliminating, substantially reducing, or at leastpartially reducing the level of an mRNA transcript or protein encoded bya gene family member from which the intron was derived. In a preferredaspect, a dsRNA construct does not contain exon sequences correspondingto a sufficient part of an exon to be capable of effectivelyeliminating, substantially reducing, or at least partially reducing thelevel of an mRNA transcript or protein encoded by a gene from which theexon was derived.

[0075] An intron can be any intron from a gene, whether endogenous orintroduced. Nucleic acid sequences of such introns can be derived from amultitude of sources, including, without limitation, databases such asEMBL and Genbank found at www-ebi.ac.uk/swisprot/; www-expasy.ch/;www-embl-heidelberg.de/; and www-ncbi.nlm.nih.gov. Nucleic acidsequences of such introns can also be derived, without limitation, fromsources such as the GENSCAN program found at//genes.mit.edu/GENSCAN.html. In a further embodiment, additionalintrons may be obtained by any method by which additional introns may beidentified. In a preferred embodiment, additional introns may beobtained by screening a genomic library with a probe of either knownexon or intron sequences. In another preferred embodiment, additionalintrons may be obtained by a comparison between genomic sequence andcorresponding cDNA sequence that allows identification of additionalintrons. In a more preferred embodiment, additional introns may beobtained by screening a genomic library with a probe of either knownexon or intron sequences. The gene may then be cloned and confirmed andany additional introns may be identified by a comparison between genomicsequence and cDNA sequence. Additional introns may, for example withoutlimitation, be amplified by PCR and used in an embodiment of the presentinvention.

[0076] In another preferred embodiment, an intron, such as for example,a soybean intron, may be cloned by alignment to an intron from anotherorganism, such as, for example, Arabidopsis. In this embodiment, thelocation of an intron in an Arabidopsis amino acid sequence, forexample, is identified. An amino acid sequence, from Arabidopsis forexample, may then be aligned, with, for example a soybean amino acidsequence, providing a prediction for the location of additional soybeanintrons.

[0077] In a preferred aspect, the target protein is selected from thegroup consisting of FAD2, FAD3, and FATB. In another preferred aspect,the target protein is selected from the group of genes consisting ofFAD2-1A, FAD2-1B, FAD2-2B, FAD3-1A, FAD3-1B, FAD3-1C, and FATB, or inanother aspect two or more of said genes. In a preferred aspect, wherehomology is present between or among gene family members, at least twotarget proteins from the same gene family are affected. In aparticularly preferred aspect, the target protein is both FAD2-1A andFAD2-1B. In another particularly preferred aspect, the target protein isboth FAD3-1A and FAD3-1C.

[0078] Representative sequences for FAD2-1A, FAD2-1B, FAD2-2B, FAD3-1A,FAD3-1B, FAD3-1C introns include, without limitation, those set forth inU.S. application Ser. No. 10/176,149, filed on Jun. 21, 2002; and U.S.patent application Ser. No. 09/638,508, filed Aug. 11, 2000; and U.S.Provisional Application Serial No. 60/151,224, filed Aug. 26, 1999; andU.S. Provisional Application Serial No. 60/172,128, filed Dec. 17, 1999,all of which applications are herein incorporated by reference in theirentireties including, without limitation, their accompanying sequencelistings.

[0079] Representative sequences for FATB introns include, withoutlimitation, those set forth in the present application at SEQ ID NOs:41, 42, and 43, as well as those set forth in U.S. Pat. Nos. 5,723,761,5,955,329, 5,955,650, 6,150,512, 6,331,664, and 6,380,462; andInternational Patent Publication Nos. WO 01/35726, WO 01/36598, and WO02/15675.

[0080] Representative sequences for FATB introns also include, withoutlimitation, those set forth in U.S. Provisional Application Serial No.60/390,185, filed Jun. 21, 2002.

[0081] In a preferred aspect, the target protein is encoded by onemember of a gene family. In another preferred aspect, the target gene isa member of a gene family. A particularly preferred use of the presentinvention is where two or more genes within the gene family exhibitsimilar nucleic acid sequences within a coding region for the targetprotein but exhibit dissimilar nucleic acid sequences within atranscribed intron region. In this aspect, a first nucleic acid sequenceis similar to a second nucleic acid sequence if a dsRNA molecule to thefirst nucleic acid sequence reduces the level of a protein and/or atranscript which is encoded by the second nucleic acid sequence.Likewise, in this aspect, a first nucleic acid sequence is dissimilar toa second nucleic acid sequence if a dsRNA molecule directed to the firstnucleic acid sequence does not reduce the level of a second proteinand/or a transcript which is encoded by the second nucleic acidsequence.

[0082] In a preferred aspect, the target gene or target protein is anon-viral gene or protein. In another preferred aspect, the target geneor target protein is an endogenous gene or protein. In a furtherpreferred aspect, the intron is an intron located between exons. Inanother preferred aspect, the intron is an intron that is within a 5′ or3′ UTR. In another preferred aspect, the target gene or protein is anon-endogenous gene or protein; for example, the target gene or proteinmay be found in a plant pest, such as, for example, in a plant nematode.

[0083] Further preferred embodiments of the invention are nucleic acidmolecules that are at least 85% identical, preferably at least 90%identical, more preferably 95, 97, 98, 99% identical, or most preferably100% identical over their entire length to an intron.

[0084] “Identity,” as is well understood in the art, is a relationshipbetween two or more polypeptide sequences or two or more nucleic acidmolecule sequences, as determined by comparing the sequences. In theart, “identity” also means the degree of sequence relatedness betweenpolypeptide or nucleic acid molecule sequences, as determined by thematch between strings of such sequences. “Identity” can be readilycalculated by known methods including, but not limited to, thosedescribed in Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York (1988); Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M. and Griffin, H. G.,eds., Humana Press, New Jersey (1994); Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press (1987); Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., Stockton Press, New York(1991); and Carillo, H., and Lipman, D., SIAM J. Applied Math, 48:1073(1988). Methods to determine identity are designed to give the largestmatch between the sequences tested. Moreover, methods to determineidentity are codified in publicly available programs.

[0085] Computer programs which can be used to determine identity betweentwo sequences include, but are not limited to, GCG (Devereux, J., etal., Nucleic Acids Research 12(1):387 (1984); suite of five BLASTprograms, three designed for nucleotide sequences queries (BLASTN,BLASTX, and TBLASTX) and two designed for protein sequence queries(BLASTP and TBLASTN) (Coulson, Trends in Biotechnology, 12:76-80 (1994);Birren et al., Genome Analysis, 1:543-559 (1997)). The BLASTX program ispublicly available from NCBI and other sources (BLAST Manual, Altschul,S., et al., NCBI NLM NIH, Bethesda, Md. 20894; Altschul, S., et al., J.Mol. Biol., 215:403-410 (1990)). The well-known Smith Waterman algorithmcan also be used to determine identity.

[0086] Parameters for polypeptide sequence comparison typically includethe following:

[0087] Algorithm: Needleman and Wunsch, J. Mol. Biol., 48:443-453 (1970)

[0088] Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc.Natl. Acad. Sci. USA, 89:10915-10919 (1992)

[0089] Gap Penalty: 12

[0090] Gap Length Penalty: 4

[0091] A program which can be used with these parameters is publiclyavailable as the “gap” program from Genetics Computer Group, Madison,Wis. The above parameters along with no penalty for end gap are thedefault parameters for peptide comparisons.

[0092] Parameters for nucleic acid molecule sequence comparison includethe following:

[0093] Algorithm: Needleman and Wunsch, J. Mol. Bio., 48:443-453 (1970)

[0094] Comparison matrix: matches−+10; mismatches=0

[0095] Gap Penalty: 50

[0096] Gap Length Penalty: 3

[0097] As used herein, “% identity” is determined using the aboveparameters as the default parameters for nucleic acid molecule sequencecomparisons and the “gap” program from GCG, version 10.2.

[0098] The invention further relates to nucleic acid molecules thathybridize to a plant intron. In particular, the invention relates tonucleic acid molecules that hybridize under stringent conditions to theabove-described nucleic acid molecules. As used herein, the terms“stringent conditions” and “stringent hybridization conditions” meanthat hybridization will generally occur if there is at least 95% andpreferably at least 97% identity between the sequences. An example ofstringent hybridization conditions is overnight incubation at 42° C. ina solution comprising 50% formamide, 5×SSC (150 mM NaCl, 15 mM trisodiumcitrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10%dextran sulfate, and 20 micrograms/milliliter denatured, sheared salmonsperm DNA, followed by washing the hybridization support in 0.1×SSC atapproximately 65° C. Other hybridization and wash conditions are wellknown and are exemplified in Sambrook et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor, N.Y. (1989),particularly Chapter 11. As used herein, two nucleic acid molecules aresaid to be capable of specifically hybridizing to one another if the twomolecules are capable of forming an anti-parallel, double-strandednucleic acid structure.

[0099] One subset of the nucleic acid molecules of the inventionincludes fragment nucleic acid molecules. For example, fragment nucleicacid molecules may consist of significant portion(s) of, or indeed mostof, a plant intron. Alternatively, fragments may comprise smalleroligonucleotides having from about 15 to about 400 contiguous nucleotideresidues and more preferably, about 15 to about 45 contiguous nucleotideresidues, about 20 to about 45 contiguous nucleotide residues, about 15to about 30 contiguous nucleotide residues, about 21 to about 30contiguous nucleotide residues, about 21 to about 25 contiguousnucleotide residues, about 21 to about 24 contiguous nucleotideresidues, about 19 to about 25 contiguous nucleotide residues, or about21 contiguous nucleotides. In a preferred embodiment, a fragment shows100% identity to the plant intron. In another preferred embodiment, afragment comprises a portion of a larger nucleic acid sequence.

[0100] In another aspect, a fragment nucleic acid molecule has a nucleicacid sequence that is at least 15, 25, 50, or 100 contiguous nucleotidesof a nucleic acid molecule of the present invention. In a preferredembodiment, a nucleic acid molecule has a nucleic acid sequence that isat least 15, 25, 50, or 100 contiguous nucleotides of a plant intron.

[0101] In one aspect of the present invention the nucleic acids of thepresent invention are said to be introduced nucleic acid molecules. Anucleic acid molecule is said to be “introduced” if it is inserted intoa cell or organism as a result of human manipulation, no matter howindirect. Examples of introduced nucleic acid molecules include, but arenot limited to, nucleic acids that have been introduced into cells viatransformation, transfection, injection, and projection, and those thathave been introduced into an organism via methods including, but notlimited to, conjugation, endocytosis, and phagocytosis. The cell ororganism can be, or can be derived from, a plant, plant cell, algae,algae cell, fungus, fungal cell, or bacterial cell. A nucleic acidmolecule of the present invention may be stably integrated into anuclear, chloroplast or mitochondrial genome, preferably into thenuclear genome.

[0102] An agent, preferably a dsRNA molecule, is preferably capable ofproviding at least a partial reduction, more preferably a substantialreduction, or most preferably effective elimination of another agentsuch as a protein or mRNA.

[0103] As used herein, “a reduction” of the level of an agent such as aprotein or mRNA means that the level is reduced relative to a cell ororganism lacking a dsRNA molecule capable of reducing the agent.

[0104] As used herein, “at least a partial reduction” of the level of anagent such as a protein or mRNA means that the level is reduced at least25% relative to a cell or organism lacking a dsRNA molecule capable ofreducing the agent.

[0105] As used herein, “a substantial reduction” of the level of anagent such as a protein or mRNA means that the level is reduced relativeto a cell or organism lacking a dsRNA molecule capable of reducing theagent, where the reduction of the level of the agent is at least 75%.

[0106] As used herein, “an effective elimination” of an agent such as aprotein or mRNA is relative to a cell or organism lacking a dsRNAmolecule capable of reducing the agent, where the reduction of the levelof the agent is greater than 95%.

[0107] An agent, preferably a dsRNA molecule, is preferably capable ofproviding at least a partial reduction, more preferably a substantialreduction, or most preferably effective elimination of another agentsuch as a protein or mRNA, wherein the agent leaves the level of asecond agent essentially unaffected, substantially unaffected, orpartially unaffected.

[0108] As used herein, “essentially unaffected” refers to a level of anagent such as a protein or mRNA transcript that is either not altered bya particular event or altered only to an extent that does not affect thephysiological function of that agent. In a preferred aspect, the levelof the agent that is essentially unaffected is within 20%, morepreferably within 10%, and even more preferably within 5% of the levelat which it is found in a cell or organism that lacks a nucleic acidmolecule capable of selectively reducing another agent.

[0109] As used herein, “substantially unaffected” refers to a level ofan agent such as a protein or mRNA transcript in which the level of theagent that is substantially unaffected is within 49%, more preferablywithin 35%, and even more preferably within 24% of the level at which itis found in a cell or organism that lacks a nucleic acid moleculecapable of selectively reducing another agent.

[0110] As used herein, “partially unaffected” refers to a level of anagent such as a protein or mRNA transcript in which the level of theagent that is partially unaffected is within 80%, more preferably within65%, and even more preferably within 50% of the level at which it isfound in a cell or organism that lacks a nucleic acid molecule capableof selectively reducing another agent.

[0111] When levels of an agent are compared, such a comparison ispreferably carried out between organisms with a similar geneticbackground. In another even more preferable aspect, a similar geneticbackground is a background where the organisms being compared areplants, and the plants are isogenic except for any genetic materialoriginally introduced using plant transformation techniques.

[0112] In a preferred aspect, the capability of a nucleic acid moleculeto reduce or selectively reduce the level of a gene relative to anothergene is carried out by a comparison of levels of mRNA transcripts. Asused herein, mRNA transcripts include processed and non-processed mRNAtranscripts. In another preferred aspect, the capability of a nucleicacid molecule to reduce or selectively reduce the level of a generelative to another gene is carried out by a comparison of phenotype. Ina preferred aspect, the comparison of phenotype is a comparison of oilcomposition.

[0113] In a further embodiment, a nucleic acid molecule, when introducedinto a cell or organism, selectively reducing the level of a proteinand/or transcript encoded by a first gene while leaving the level of aprotein and/or transcript encoded by a second gene partially unaffected,substantially unaffected, or essentially unaffected, also alters the oilcomposition of the cell or organism.

[0114] Organisms

[0115] The constructs of this invention can be used to suppress any genecontaining unique intron sequence of a target gene for suppression in aeukaryotic organism, such as for example without limitation, plants oranimals, such as mammals, insects, nematodes, fish, and birds. Thetarget gene for suppression can be an endogenous gene or a transgene inan organism to be transformed with a construct of the present invention.Alternatively, the target gene for suppression can be in anon-transgenic organism which acquires the dsRNA or DNA producing dsRNAby ingestion or infection by a transgenic organism. See e.g., U.S. Pat.No. 6,506,559.

[0116] Thus, an aspect of this invention provides a method where thetarget gene for suppression encodes a protein in an insect or nematodewhich is a pest to a plant. In an aspect, a method comprises introducinginto the genome of a pest-targeted plant a nucleic acid constructcomprising DNA which is transcribed into RNA that forms at least onedouble-stranded RNA molecule which is effective for reducing expressionof a target gene within the pest when the pest, e.g., insect or nematodeingests cells from said plant. In a preferred embodiment, the genesuppression is fatal to the pest.

[0117] Plant Constructs and Plant Transformants

[0118] Exogenous genetic material may be transferred into a plant celland the plant cell regenerated into a whole, fertile or sterile plant orplant part. Exogenous genetic material is any genetic material, whethernaturally occurring or otherwise, from any source that is capable ofbeing inserted into any organism. Such exogenous genetic materialincludes, without limitation, nucleic acid molecules that encode a dsRNAmolecule of the present invention.

[0119] In a preferred aspect, a plant cell or plant of the presentinvention includes a nucleic acid molecule that exhibits sufficienthomology to one or more plant introns such that when it is expressed asa dsRNA construct, it is capable of effectively eliminating,substantially reducing, or at least partially reducing the level of anmRNA transcript or protein encoded by the gene from which the intron wasderived or any gene which has an intron with homology to the targetintron.

[0120] In one embodiment of the invention, the expression level of aprotein or transcript in one family member of that gene is selectivelyreduced while leaving the level of a protein or transcript of a secondfamily member partially unaffected. In a preferred embodiment of theinvention, the expression level of a protein or transcript in one familymember of that gene is selectively reduced while leaving the level of aprotein or transcript of a second family member substantiallyunaffected. In a highly preferred embodiment of the invention, theexpression level of a protein or transcript in one family member of thatgene is selectively reduced while leaving the level of a protein ortranscript of a second family member essentially unaffected.

[0121] In a particularly preferred embodiment, a transgenic plantincludes a nucleic acid molecule that comprises a nucleic acid sequence,which is capable of selectively reducing the expression level of aprotein and/or transcript encoded by certain FAD2 and/or FAD3 geneswhile leaving the level of a protein and/or transcript of at least oneother FAD2 or FAD3 gene in the plant partially unaffected or morepreferably substantially or essentially unaffected.

[0122] The levels of target products such as transcripts or proteins maybe decreased throughout an organism such as a plant or mammal, or suchdecrease in target products may be localized in one or more specificorgans or tissues of the organism. For example, the levels of productsmay be decreased in one or more of the tissues and organs of a plantincluding without limitation: roots, tubers, stems, leaves, stalks,fruit, berries, nuts, bark, pods, seeds and flowers. A preferred organis a seed.

[0123] The present invention provides nucleic acid constructs thatencode a dsRNA molecule of the present invention. In a preferred aspect,such constructs comprise at least one sequence that when transcribed isa sense sequence that exhibits sufficient identity to an intron whichwhen expressed in the presence of its complement (antisense) forms adouble-stranded RNA molecule capable of at least partially reducing thelevel of an mRNA containing the intron sequence. In another preferredaspect, such constructs comprise at least one sequence that whentranscribed is a sense sequence that exhibits sufficient identity tomore than one intron, preferably more than two introns, more preferablymore than three introns, which when expressed in the presence of theircomplements (antisense) forms a double-stranded RNA molecule capable ofat least partially reducing the level of all mRNAs containing the intronsequence.

[0124] In one aspect, e.g. for suppressing plant genes, the nucleic acidconstruct comprises a plant promoter and a DNA sequence capable ofexpressing a first RNA that exhibits identity to a transcribed intron ofa plant gene and expressing a second RNA capable of forming adouble-stranded RNA molecule with said first RNA. In a preferred aspect,the first RNA exhibits identity to at least two, more preferably atleast three or at least four, five or six plant introns. In anotherpreferred aspect, the first RNA and the second RNA are encoded byphysically linked nucleic acid sequences.

[0125] When physically linked, the nucleic acid sequences which encodethe first RNA and the second RNA (the complement of the first RNA) canin a preferred aspect be separated by a sequence (spacer sequence),preferably one that promotes the formation of a dsRNA molecule. Examplesof such sequences include those set forth in Wesley et al., supra, andHamilton et al., Plant J., 15:737-746 (1988) which are capable offorming a hairpin loop between hybridized RNA. In a preferred aspect,the separating sequence is a spliceable intron. Spliceable intronsinclude, but are not limited to, an intron selected from the groupconsisting of Pdk intron, FAD3 intron #5, FAD3 intron #1, FAD3 intron#3A, FAD3 intron #3B, FAD3 intron #3C, FAD3 intron #4, FAD3 intron #5,FAD2 intron #1, FAD2-2 intron. Preferred spliceable introns include, butare not limited to, an intron selected from the group consisting of FAD3intron #1, FAD3 intron #3A, FAD3 intron #3B, FAD3 intron #3C, and FAD3intron #5. Other preferred spliceable introns include, but are notlimited to, a spliceable intron that is about 0.75 kb to about 1.1 kb inlength and is capable of facilitating an RNA hairpin structure. Onenon-limiting example of a particularly preferred spliceable intron isFAD3 intron #5.

[0126] In a particularly preferred aspect, the construct comprises anucleic acid where a first RNA exhibits identity to two or more,preferably three or more introns where the introns are selected from thegroup consisting of FAD2-1A, FAD2-1B, FAD2-2B, FAD3-1A, FAD3-1B,FAD3-1C, and FATB introns.

[0127] Constructs may be designed, without limitation, in a 7Sexpression cassette such as the pCGN3892 vector (FIG. 1). Particularlypreferred constructs include the following pCGN3892 derived constructs:(1) 7S promoter—FAD2-1A sense intron—FAD3-1C sense intron—FAD3-1A senseintron FAD3-1B sense intron—spliceable FAD3 intron #5—FAD3-1B antisenseintron—FAD3-1A antisense intron—FAD3-1C antisense intron—FAD2-1Aantisense intron—pea rbcS; (2) 7S promoter—FAD2-1A sense intron—FAD3-1Asense intron—FAD3-1B sense intron—spliceable FAD3 intron #5—FAD3-1Bantisense intron—FAD3-1A antisense intron—FAD2-1A antisense intron—pearbcS; (3) 7S promoter—FAD2-1A sense intron—FAD3-1A senseintron—spliceable FAD3 intron #5—FAD3-1A antisense intron—FAD2-1Aantisense intron—pea rbcS; (4) 7S promoter—FAD2-1A senseintron—spliceable FAD3 intron #5—FAD2-1A antisense intron—pea rbcS; (5)7S promoter—FAD3-1A sense intron—spliceable FAD3 intron #5—FAD3-1Aantisense intron—pea rbcS; (6) 7S promoter—FAD2-1A sense intron—FAD3-1Asense 3′UTR—spliceable FAD3 intron #5—FAD3-1A antisense 3′UTR—FAD2-1Aantisense intron—pea rbcS; and (7) 7S promoter—FAD2-1A senseintron—FAD3-1A sense 3′UTR—FAD3-1B sense 3′UTR—spliceable FAD3 intron#5—FAD3-1B antisense 3′UTR—FAD3-1A antisense 3′UTR—FAD2-1A antisenseintron—pea rbcS.

[0128] Other preferred constructs may be prepared using one or more FATBintrons in a 7S expression cassette such as the pCGN3892 vector (FIG.1). For example, other particularly preferred constructs include withoutlimitation the following pCGN3892 derived constructs: (1) 7Spromoter—FATB sense intron I—FATB sense intron II—spliceable FAD3 intron#5—FATB antisense intron II—FATB antisense intron I—pea rbcS; (2) 7Spromoter—FATB sense intron II—FATB sense intron I—spliceable FAD3 intron#5—FATB antisense intron I—FATB antisense intron II—pea rbcS; (3) 7Spromoter—FATB sense intron—spliceable FAD3 intron #5—FATB antisenseintron—pea rbcS.

[0129] In another embodiment of the present invention, a constructlacking a promoter and a 3′ flanking region may be injected directlyinto either the cytoplasm, or preferably into the nucleus, of a cell viamicroinjection.

[0130] Transgenic DNA constructs used for transforming plant cells forintron-based RNAi will comprise the heterologous DNA which encodes thedouble-stranded RNA and a promoter to express the heterologous DNA inthe host plant cells. As is well known in the art, such constructstypically also comprise a promoter and other regulatory elements, 3′untranslated regions (such as polyadenylation sites), transit or signalpeptides and marker genes elements as desired. For instance, see U.S.Pat. Nos. 5,858,642 and 5,322,938 which disclose versions of theconstitutive promoter derived from cauliflower mosaic virus (CaMV35S),U.S. Pat. No. 6,437,217 which discloses a maize RS81 promoter, U.S. Pat.No. 5,641,876 which discloses a rice actin promoter, U.S. Pat. No.6,426,446 which discloses a maize RS324 promoter, U.S. Pat. No.6,429,362 which discloses a maize PR-1 promoter, U.S. Pat. No. 6,232,526which discloses a maize A3 promoter, U.S. Pat. No. 6,177,611 whichdiscloses constitutive maize promoters, U.S. Pat. No. 6,433,252 whichdiscloses a maize L3 oleosin promoter, U.S. Pat. No. 6,429,357 whichdiscloses a rice actin 2 promoter and intron, U.S. Pat. No. 5,837,848which discloses a root specific promoter, U.S. Pat. No. 6,084,089 whichdiscloses cold-inducible promoters, U.S. Pat. No. 6,294,714 whichdiscloses light-inducible promoters, U.S. Pat. No. 6,140,078 whichdiscloses salt-inducible promoters, U.S. Pat. No. 6,252,138 whichdiscloses pathogen-inducible promoters, U.S. Pat. No. 6,175,060 whichdiscloses phosphorus deficiency-inducible promoters, U.S. PatentApplication Publication 2002/0192813A1 which discloses 5′, 3′ and intronelements useful in the design of effective plant expression vectors,U.S. patent application Ser. No. 09/078,972 which discloses a coixinpromoter, U.S. patent application Ser. No. 09/757,089 which discloses amaize chloroplast aldolase promoter, all of which are incorporatedherein by reference.

[0131] Constructs or vectors may also include, with the region ofinterest, a nucleic acid sequence that acts, in whole or in part, toterminate transcription of that region. A number of such sequences havebeen isolated, including the Tr7 3′ sequence and the NOS 3′ sequence(Ingelbrecht et al., The Plant Cell 1:671-680 (1989); Bevan et al.,Nucleic Acids Res. 11:369-385 (1983)). Regulatory transcript terminationregions can be provided in plant expression constructs of this inventionas well. Transcript termination regions can be provided by the DNAsequence encoding the gene of interest or a convenient transcriptiontermination region derived from a different gene source, for example,the transcript termination region that is naturally associated with thetranscript initiation region. The skilled artisan will recognize thatany convenient transcript termination region that is capable ofterminating transcription in a plant cell can be employed in theconstructs of the present invention.

[0132] A vector or construct may also include regulatory elements.Examples of such include the Adh intron 1 (Callis et al., Genes andDevelop. 1:1183-1200 (1987)), the sucrose synthase intron (Vasil et al.,Plant Physiol. 91:1575-1579 (1989)) and the TMV omega element (Gallie etal., The Plant Cell 1:301-311 (1989)). These and other regulatoryelements may be included when appropriate.

[0133] In practice DNA is introduced into only a small percentage oftarget cells in any one experiment. Marker genes are used to provide anefficient system for identification of those cells that are stablytransformed by receiving and integrating a transgenic DNA construct intotheir genomes. Preferred marker genes provide selective markers whichconfer resistance to a selective agent, such as an antibiotic orherbicide. Potentially transformed cells are exposed to the selectiveagent. In the population of surviving cells will be those cells where,generally, the resistance-conferring gene has been integrated andexpressed at sufficient levels to permit cell survival. Cells may betested further to confirm stable integration of exogenous DNA. Usefulselective marker genes include those conferring resistance toantibiotics such as kanamycin (nptII), hygromycin B (aph IV) andgentamycin (aac3 and aacC4) or resistance to herbicides such asglufosinate (bar or pat) and glyphosate (EPSPS). Examples of suchselectable markers are illustrated in U.S. Pat. Nos. 5,550,318;5,633,435; 5,780,708 and 6,118,047, all of which are incorporated hereinby reference. Screenable markers which provide an ability to visuallyidentify transformants can also be employed, e.g., a gene expressing acolored or fluorescent protein such as a luciferase or green fluorescentprotein (GFP) or a gene expressing a beta-glucuronidase or uidA gene(GUS) for which various chromogenic substrates are known.

[0134] Transformation Methods and Transgenic Plants

[0135] Methods and compositions for transforming plants by introducing atransgenic DNA construct or a nucleic acid molecule of the presentinvention into a plant genome in the practice of this invention caninclude any of the well-known and demonstrated methods. Preferredmethods of plant transformation are microprojectile bombardment asillustrated in U.S. Pat. Nos. 5,015,580; 5,550,318; 5,538,880;6,160,208; 6,399,861 and 6,403,865 and Agrobacterium-mediatedtransformation as illustrated in U.S. Pat. Nos. 5,635,055; 5,824,877;5,591,616; 5,981,840 and 6,384,301, all of which are incorporated hereinby reference. See also U.S. patent application Ser. No. 09/823,676,incorporated herein by reference, for a description of vectors,transformation methods, and production of transformed Arabidopsisthaliana plants where transcription factors such as G1073 areconstitutively expressed by a CaMV35S promoter.

[0136] Transformation methods of this invention to provide plants withenhanced environmental stress tolerance are preferably practiced intissue culture on media and in a controlled environment. “Media” refersto the numerous nutrient mixtures that are used to grow cells in vitro,that is, outside of the intact living organism. Recipient cell targetsinclude, but are not limited to, meristem cells, Type I, Type II, andType III callus, immature embryos and gametic cells such as microspores,pollen, sperm and egg cells. It is contemplated that any cell from whicha fertile plant may be regenerated is useful as a recipient cell. Callusmay be initiated from tissue sources including, but not limited to,immature embryos, seedling apical meristems, microspores and the like.Those cells, which are capable of proliferating as calli, also arerecipient cells for genetic transformation. Practical transformationmethods and materials for making transgenic plants of this invention,e.g. various media and recipient target cells, transformation ofimmature embryos and subsequent regeneration of fertile transgenicplants are disclosed in U.S. Pat. No. 6,194,636 and U.S. patentapplication Ser. No. 09/757,089, which are incorporated herein byreference.

[0137] Examples of species that have been transformed by microprojectilebombardment include monocot species such as maize (PCT Publication WO95/06128), barley, wheat (U.S. Pat. No. 5,563,055, specificallyincorporated herein by reference in its entirety), rice, oat, rye,sugarcane, and sorghum; as well as a number of dicots including tobacco,soybean (U.S. Pat. No. 5,322,783, specifically incorporated herein byreference in its entirety), sunflower, peanut, cotton, tomato, andlegumes in general (U.S. Pat. No. 5,563,055, specifically incorporatedherein by reference in its entirety).

[0138] The regeneration, development, and cultivation of plants fromvarious transformed explants is well documented in the art. Thisregeneration and growth process typically includes the steps ofselecting transformed cells and culturing those individualized cellsthrough the usual stages of embryonic development through the rootedplantlet stage. Transgenic embryos and seeds are similarly regenerated.The resulting transgenic rooted shoots are thereafter planted in anappropriate plant growth medium such as soil. Cells that survive theexposure to the selective agent, or cells that have been scored positivein a screening assay, may be cultured in media that supportsregeneration of plants. Developing plantlets are transferred to soilless plant growth mix, and hardened off, prior to transfer to agreenhouse or growth chamber for maturation.

[0139] The present invention can be used with any transformable cell ortissue. Those of skill in the art recognize that a number of plant cellsor tissues are transformable in which after insertion of exogenous DNAand appropriate culture conditions the plant cells or tissues can forminto a differentiated plant. Tissue suitable for these purposes caninclude but is not limited to immature embryos, scutellar tissue,suspension cell cultures, immature inflorescence, shoot meristem, nodalexplants, callus tissue, hypocotyl tissue, cotyledons, roots, andleaves.

[0140] Any suitable plant culture medium can be used. Examples ofsuitable media would include but are not limited to MS-based media(Murashige and Skoog, Physiol. Plant, 15:473-497, (1962) or N6-basedmedia (Chu et al., Scientia Sinica 18:659, (1975) supplemented withadditional plant growth regulators including but not limited to auxins,cytokinins, ABA, and gibberellins. Those of skill in the art arefamiliar with the variety of tissue culture media, which whensupplemented appropriately, support plant tissue growth and developmentand are suitable for plant transformation and regeneration. These tissueculture media can either be purchased as a commercial preparation, orcustom prepared and modified. Those of skill in the art are aware thatmedia and media supplements such as nutrients and growth regulators foruse in transformation and regeneration and other culture conditions suchas light intensity during incubation, pH, and incubation temperaturescan be optimized for the particular variety of interest.

[0141] Any of the nucleic acid molecules of the invention may beintroduced into a plant cell in a permanent or transient manner incombination with other genetic elements, for example, including but notlimited to, vectors, promoters, and enhancers. Further, any of thenucleic acid molecules of the invention may be introduced into a plantcell in a manner that allows for expression or overexpression of theprotein or fragment thereof encoded by the nucleic acid molecule.

[0142] It is understood that two or more nucleic molecules of thepresent invention may be introduced into a plant using a singleconstruct and that construct can contain more than one promoter. Inembodiments where the construct is designed to express two nucleic acidmolecules, it is preferred that the two promoters are (i) twoconstitutive promoters, (ii) two seed-specific promoters, or (iii) oneconstitutive promoter and one seed-specific promoter. Preferredseed-specific and constitutive promoters are a napin and a 7S promoter,respectively. It is understood that two or more of the nucleic moleculesmay be physically linked and expressed utilizing a single promoter,preferably a seed-specific or constitutive promoter.

[0143] It is further understood that two or more nucleic acids of thepresent invention may be introduced into a plant using two or moredifferent constructs. Alternatively, two or more nucleic acids of thepresent invention may be introduced into two different plants and theplants may be crossed to generate a single plant expressing two or morenucleic acids. In an RNAi embodiment, it is understood that the senseand antisense strands may be introduced into the same plant on oneconstruct or two constructs. Alternatively, the sense and antisensestrands may be introduced into two different plants and the plants maybe crossed to generate a single plant expressing both sense andantisense strands.

[0144] The present invention also provides for parts of the plants,particularly reproductive or storage parts. Plant parts, withoutlimitation, include seed, endosperm, ovule, pollen, roots, tubers,stems, leaves, stalks, fruit, berries, nuts, bark, pods, seeds andflowers. In a particularly preferred embodiment of the presentinvention, the plant part is a seed.

[0145] The present invention also provides a container of over 10,000,more preferably 20,000, and even more preferably 40,000 seeds where over10%, more preferably 25%, more preferably 50% and even more preferably75% or 90% of the seeds are seeds derived from a plant of the presentinvention.

[0146] The present invention also provides a container of over 10 kg,more preferably 25 kg, and even more preferably 50 kg seeds where over10%, more preferably 25%, more preferably 50% and even more preferably75% or 90% of the seeds are seeds derived from a plant of the presentinvention.

[0147] Plants of the present invention can be part of or generated froma breeding program. The choice of breeding method depends on the mode ofplant reproduction, the heritability of the trait or traits beingimproved, and the type of cultivar used commercially (e.g., F₁ hybridcultivar, pureline cultivar, etc). Selected, non-limiting approaches,for breeding the plants of the present invention are set forth below. Abreeding program can be enhanced using marker-assisted selection of theprogeny of any cross. It is further understood that any commercial andnon-commercial cultivars can be utilized in a breeding program. Factorssuch as, for example, emergence vigor, vegetative vigor, stresstolerance, disease resistance, branching, flowering, seed set, seedsize, seed density, standability, and threshability will generallydictate the choice.

[0148] For highly heritable traits, a choice of superior individualplants evaluated at a single location will be effective, whereas fortraits with low heritability, selection should be based on mean valuesobtained from replicated evaluations of families of related plants.Popular selection methods commonly include pedigree selection, modifiedpedigree selection, mass selection, and recurrent selection. In apreferred embodiment, a backcross or recurrent breeding program isundertaken.

[0149] The complexity of inheritance influences choice of the breedingmethod. Backcross breeding can be used to transfer one or a fewfavorable genes for a highly heritable trait into a desirable cultivar.This approach has been used extensively for breeding disease-resistantcultivars. Various recurrent selection techniques are used to improvequantitatively inherited traits controlled by numerous genes. The use ofrecurrent selection in self-pollinating crops depends on the ease ofpollination, the frequency of successful hybrids from each pollination,and the number of hybrid offspring from each successful cross.

[0150] Breeding lines can be tested and compared to appropriatestandards in environments representative of the commercial targetarea(s) for two or more generations. The best lines are candidates fornew commercial cultivars; those still deficient in traits may be used asparents to produce new populations for further selection.

[0151] One method of identifying a superior plant is to observe itsperformance relative to other experimental plants and to a widely grownstandard cultivar. If a single observation is inconclusive, replicatedobservations can provide a better estimate of genetic worth. A breedercan select and cross two or more parental lines, followed by repeatedselfing and selection, producing many new genetic combinations.

[0152] The development of new cultivars requires the development andselection of varieties, the crossing of these varieties and theselection of superior hybrid crosses. The hybrid seed can be produced bymanual crosses between selected male-fertile parents or by using malesterility systems. Hybrids are selected for certain single gene traitssuch as pod color, flower color, seed yield, pubescence color, orherbicide resistance, which indicate that the seed is truly a hybrid.Additional data on parental lines, as well as the phenotype of thehybrid, influence a breeder's decision whether to continue with thespecific hybrid cross.

[0153] Agents of the present invention can be utilized in a variety ofmethods. For example, the present invention provides a method ofaltering the expression of a target gene comprising (a) introducing intoa cell a first DNA sequence capable of expressing a first RNA whichexhibits identity to a transcribed intron of the target gene and asecond DNA sequence and a method of modifying a level of a targetprotein comprising: (a) growing a plant having integrated into a genomea nucleic acid molecule comprising a first DNA sequence which encodes afirst RNA that exhibits identity to a transcribed intron of an mRNA thatencodes the target protein and a second DNA sequence capable ofexpressing a second RNA capable of forming a double-stranded RNAmolecule with the first RNA and (b) expressing the first and second RNA.In a preferred aspect, the expression of a target gene is altered ormodified if the level of an mRNA or protein encoded by that gene isaltered, in a more preferred aspect, a method of the present inventionprovides for at least a partial reduction, or more preferably asubstantial reduction or effective elimination of an encoded agent suchas a protein or mRNA.

[0154] The following examples are illustrative and not intended to belimiting in any way.

EXAMPLES Example 1 This Example Illustrates the Identification ofIntrons Which are Useful for Demonstrating the Suppression of GenesUsing Intron Double-Stranded RNA Molecules

[0155] 1A. Soybean A12 Desalurase (FAD2-1)

[0156] A soybean FAD2-1A sequence is identified by screening a soybeangenomic library using a soybean FAD2-1 cDNA probe. Three putative soyFAD2-1 clones are identified and plaque purified. Two of the three soyFAD2-1 clones are ligated into pBluescript II KS+ (Stratagene) andsequenced. Both clones (14-1 and 11-12) are the same and match the soyFAD2-1 cDNA exactly. A sequence of the entire FAD2-1A clone is providedin SEQ ID NO:15.

[0157] Prior to obtaining a full length clone, a portion of the FAD2-1Agenomic clone is PCR amplified using PCR primers designed from the 5′untranslated sequence (Primer 12506, 5′-ATACAA GCCACTAGGCAT-3′, SEQ IDNO:16) and within the cDNA (Primer 11698:5′-GATTGGCCATGCAATGAGGGAAAAGG-3′, SEQ ID NO:17). The resulting PCRproduct is cloned into the vector pCR 2.1 (Invitrogen) and sequenced. Asoy FAD2-1A partial genomic clone (SEQ ID NO:18) with an intron region(SEQ ID NO:1) is identified by comparison to the soybean cDNA sequenceusing the Pustell comparison program in Macvector. The FAD2-1A intron #1sequence (SEQ ID NO:1) begins after the ATG start codon, and is 420bases long.

[0158] A second FAD2-1 gene family member is also identified and cloned,and is referred to herein as FAD2-1B. The soy FAD2-1B partial genomicclone (SEQ ID NO:19) has a coding region (base pairs 1783-1785 and2191-2463) and an intron region (base pairs 1786-2190) which areidentified by comparison to the soybean cDNA sequence using the Pustellcomparison program in Macvector. The FAD2-1B intron #1 sequence (SEQ IDNO:2) begins after the ATG start codon and is 405 bases long. Otherregions in the FAD2-1B partial genomic clone (SEQ ID NO: 19) include apromoter (base pairs 1-1704) (SEQ ID NO: 22) and 5′UTR (base pairs1705-1782).

[0159] 1B. Soybean A15 Desaturase (FAD3)

[0160] A partial soybean FAD3-1A genomic sequence is PCR amplified fromsoybean DNA using primers 10632,5′-CUACUACUACUACTCGAGACAAAGCCTTTAGCCTATG-3′ (SEQ ID NO: 20), and 10633:5′-CAUCAUCAUCAUGGATCCCATGTCTCTCTATGCAAG-3′ (SEQ ID NO: 21). The ExpandLong Template PCR system (Roche Applied Sciences, Indianapolis) is usedaccording to the manufacturer's directions. The resulting PCR productsare cloned into the vector pCR 2.1 (Invitrogen) and sequenced. A soyFAD3-1A partial genomic clone sequence (SEQ ID NO: 23) and intronregions are confirmed by comparisons to the soybean FAD3-1A cDNAsequence using the Pustell program in Macvector.

[0161] From the identified partial genomic soybean FAD3-1A sequence (SEQID NO:23), seven introns are identified: FAD3-1A intron #1 (SEQ IDNO:5), FAD3-1A intron #2 (SEQ ID NO:6), FAD3-1A intron #3A (SEQ IDNO:7), FAD3-1A intron #4 (SEQ ID NO:8), FAD3-1A intron #5 (SEQ ID NO:9),FAD3-1A intron #3B (SEQ ID NO:10), and FAD3-1A intron #3C (SEQ IDNO:11). FAD3-1A intron #1 is 191 base pairs long and is located betweenpositions 294 and 484, FAD3-1A intron #2 is 346 base pairs long and islocated between positions 577 and 922, FAD3-1A intron #3A is 142 basepairs long and is located between positions 991 and 1132, FAD3-1A intron#3B is 98 base pairs long and is located between positions 1224 and1321, FAD3-1A intron #3C is 115 base pairs long and is located betweenpositions 1509 and 1623, FAD3-1A intron #4 is 1228 base pairs long andis located between positions 1707 and 2934, and FAD3-1A intron #5 is 625base pairs long and is located between positions 3075 and 3699.

[0162] Introns #3C and #4 are also PCR amplified from a second FAD3 genefamily member (FAD3-1B). Soybean FAD3-1B introns #3C and #4 are PCRamplified from soybean DNA using the following primers,5′CATGCTTTCTGTGCTTCTC 3′ (SEQ ID NO: 26) and 5′ GTTGATCCAACCATAGTCG 3′(SEQ ID NO: 27). The PCR products are cloned into the vector pCR 2.1(Invitrogen) and sequenced. Sequences for the FAD3-1B introns #3C and #4are provided in SEQ ID NOs:12 and 13, respectively.

[0163] 1C. FATB Thioesterase

[0164] A soybean FATB sequence is identified by screening a soybeangenomic library using a soybean FATB cDNA probe (SEQ ID NO: 55). Leaftissue is obtained from Asgrow soy variety A3244, ground up in liquidnitrogen and stored at −80° C. until use. 6 ml of SDS Extraction buffer(650 ml sterile ddH₂O, 100 ml 1M Tris-Cl pH 8, 100 ml 0.25M EDTA, 50 ml20% SDS, 100 ml 5M NaCl, 4 μl beta-mercaptoethanol) is added to samplesof 2 ml frozen/ground leaf tissue, and the mixture is incubated at 65°C. for 45 min. The samples are shaken every 15 min. 2 ml ice-cold 5Mpotassium acetate is added to each sample, the samples are shaken, andthen incubated on ice for 20 min. 3 ml CHCl₃ is added to each sample,and then the samples are shaken for 10 min.

[0165] The samples are then centrifuged at 10,000 rpm for 20 min, andthe protocol is continued with the supernatant. 2 ml isopropanol isadded to each sample and mixed. The samples are then centrifuged at10,000 rpm for 20 min, and the supernatant is drained. The pellet isresuspended in 200 μl RNase, and incubated at 65° C. for 20 minutes. 300μl ammonium acetate/isopropanol (1:7) is added, and mixed. The samplesare then centrifuged at 10,000 rpm for 15 minutes, and the supernatantis discarded. The pellet is rinsed with 500 l 80% ethanol, and allowedto air dry. The pellet is then resuspended in 200 μl T10E1 (10 mM Tris:1 mM EDTA). Approximately 840 μg of clean gDNA is obtained.

[0166] Based on the FATB cDNA sequence and restriction enzyme patterns,six oligonucleotides are synthesized: F1 (SEQ ID NO: 46), F2 (SEQ ID NO:47), F3 (SEQ ID NO: 48), R1 (SEQ ID NO: 49), R2 (SEQ ID NO: 50), and R3(SEQ ID NO: 51). The oligonucleotide are used in pairs for PCRamplification of the isolated soy genomic DNA: pair 1 (F1+R1), pair 2(F1+R2), pair 3 (F1+R3), pair 4 (F2+R1), pair 5 (F2+R2), pair 6 (F2+R3),pair 7 (F3+R1), and pair 8 (F3+R2). The PCR amplification is carried outas follows: 1 cycle, 95° C. for 10 min; 40 cycles, 95° C. for 1 min, 58°C. for 30 sec, 72° C. for 55 sec; 1 cycle, 72° C. for 7 min. Threepositive fragments are obtained, specifically from primer pairs 3, 6,and 7. Each fragment is cloned into vector pCR2.1 (Invitrogen). Cloningis successful for fragment #3, which is confirmed and sequenced (SEQ IDNO: 45).

[0167] Three introns are identified in the soybean FATB gene bycomparison of the genomic sequence to the cDNA sequence: intron I (SEQID NO: 41) spans base 106 to base 214 of the genomic sequence (SEQ IDNO: 45) and is 109 bp in length; intron II (SEQ ID NO: 42) spans base289 to base 1125 of the genomic sequence (SEQ ID NO: 45) and is 837 bpin length; and intron III (SEQ ID NO: 43) spans base 1635 to base 1803of the genomic sequence (SEQ ID NO: 45) and is 169 bp in length.

Example 2 This Example Illustrates Constructs for ExpressingDouble-Stranded RNA Using Separate Promoters for the Sense and AntisenseIntrons

[0168] The FAD2-1A intron #1 sequence (SEQ ID NO: 1) is amplified viaPCR using the FAD2-1A partial genomic clone (SEQ ID NO: 18) as atemplate and primers 12701 (5′-ACGAATTCCTCGAGGTAAA TTAAATTGTGCCTGC-3′(SEQ ID NO: 24)) and 12702 (5′-GCGAGATCTATCG ATCTGTGTCAAAGTATAAAC-3′(SEQ ID NO: 25)). The resulting amplification products are cloned intothe vector pCR 2.1 (Invitrogen) and sequenced. The FAD2-1A intron isthen cloned into the expression cassette, pCGN3892 (FIG. 1), in senseand antisense orientations. The vector pCGN3892 contains the soybean 7Salpha′ promoter and a pea rbcS 3′. Both gene fusions are then separatelyligated in two sequential steps into pCGN9372, a vector that containsthe CP4 gene regulated by the FMV promoter. The resulting vector, whichcontains the FAD2-1A intron in the sense and antisense orientationdriven by two separate 7S alpha′ promoters and the FMV-CP4 geneselectable marker, is transformed into soybean via Agrobacteriumtumefaciens strain ABI using methods generally described by Martinell inU.S. Pat. No. 6,384,310 to provide transgenic soybean plants with theFAD2 gene suppressed.

[0169] Four of the seven introns identified from the soybean FAD3-1Agenomic clone are PCR amplified using the FAD3-1A partial genomic cloneas template and primers as follows: FAD3-1A intron #1, primers 12568:5′-GATCGATGCCCGGGGTAATAATTTTTGTGT-3′ (SEQ ID NO: 30) and 12569:5′-CACGCCTCGAGTGTTCAATTCAATCAATG-3′ (SEQ ID NO: 31); FAD3-1A intron #2,primers 12514: 5′-CACTCGAGTTAGTTCATACTGGCT-3′ (SEQ ID NO: 32) and 12515:5′-CGCATCGATTGCAAAATCCATCAAA-3′ (SEQ ID NO: 33); FAD3-1A intron #4,primers 10926: 5′-CUACUACUACUACTCGAGCGTAAATAGTGGGTGAACAC-3′ (SEQ ID NO:34) and 10927: 5′-CAUCAUCAUCAUCTCGAGGAATTCGTCCATTTTAGTACACC-3′ (SEQ IDNO: 35); FAD3-1A intron #5, primers 10928: 5′-CUACUACUACUACTCGAGGCGCGTACATTTTATTGCTTA-3′ (SEQ ID NO: 36) and 10929: 5′-CAUCAUCAUCAUCTCGAGGAATTCTGCAGTGAATCCAAATG-3′ (SEQ ID NO: 37). The resulting PCRproducts for each intron are cloned into the vector pCR 2.1 (Invitrogen)and sequenced.

[0170] FAD3-1A introns #1, #2, #4 and #5 are all ligated separately intothe pCGN3892, in sense and antisense orientations. pCGN3892 (FIG. 1)contains the soybean 7S alpha′ promoter and a pea rbcS 3′. These fusionsare ligated in two sequential steps into pCGN9372, a vector thatcontains the CP4 gene regulated by the FMV promoter for transformationinto soybean. The resulting vectors contain a sense and antisense copyof each intron driven by two separate 7S alpha′ promoters. For example,one such vector contains the FAD3-1A intron #1 in the sense andantisense orientation driven by two separate 7S alpha′ promoters and theFMV-CP4 gene selectable marker. A second example contains the FAD3-1Aintron #4 in the sense and antisense orientation driven by two separate7S alpha′ promoters and the FMV-CP4 gene selectable marker. Vectorscontaining such sense and antisense constructs are transformed intosoybean via Agrobacterium tumefaciens strain ABI using methods generallydescribed by Martinell in U.S. Pat. No. 6,384,310.

Example 3 This Example Illustrates Constructs for ExpressingDouble-Stranded RNA Using Separate Promoters for the Sense And AntisenseIntrons

[0171] The soybean FATB intron II sequence (SEQ ID NO: 42) is amplifiedvia PCR using the FATB fragment #3 partial genomic clone (SEQ ID NO: 45)as a template and primers 18133 (SEQ ID NO: 52) and 18134 (SEQ ID NO:53). PCR amplification is carried out as follows: 1 cycle, 95° C. for 10min; 25 cycles, 95° C. for 30 sec, 62° C. for 30 sec, 72° C. for 30 sec;1 cycle, 72° C. for 7 min.

[0172] PCR amplification results in a product (SEQ ID NO: 54) that is854 bp long, including reengineered restriction sites at both ends. TheFATB intron #2 PCR product is cloned separately in two sequential stepsdirectly into the expression cassette pCGN3892 (FIG. 1) in a sense orantisense orientation. Vector pCGN3892 contains the soybean 7Salpha′promoter and a pea RBCS 3′. The resulting vector contains a senseand antisense copy of the FATB intron #2, each of which is driven by aseparate 7S alpha′ promoter. The resulting gene expression construct, isused for transformation of soybean using Agrobacterium methods asdescribed herein.

Example 4

[0173] The following sixteen steps illustrate the construction of avector pMON68546 designed for plant transformation to suppress FAD2,FAD3, and FA TB genes in soybean. In particular, the construct comprisesa 7S alpha promoter operably linked to a series of soybeansense-oriented introns, i.e., a FAD2-1A intron #1, a FAD3-1A intron #4,a FATB intron #2, a FAD3-1B intron #4, a hairpin loop-forming spliceableintron, and a complementary series of soybean anti-sense-orientedintrons, i.e., a FAD3-1B intron #4, a FATB intron #2, a FAD3-1A intron#4 and a FAD2-1A intron #1.

[0174] Step1—The soybean FAD3-1A intron #5, which serves as thespliceable intron portion of the RNAi construct, is PCR amplified usingSoy genomic DNA as template, with the following primers:

[0175]5′primer=19037=ACTAGTATATTGAGCTCATATTCCACTGCAGTGGATATTGTTTAAACATAGCTAGCATATTACGCGTATATTATACAAGCTTATATTCCCGGGATATTGTCGACATATTAGCGGTACATTTTATTGCTTATTCAC 3′primer=19045=ACTAGTATATTGAGCTCATATTCCTGCAGGATATTCTCGAGATATTCACGGTAGTAATCTCCAAGAACTGGTTTTGCTGCTTGTGTCTGCAGTGAATC. These primers add cloningsites to the 5′ and 3′ ends. To 5′ end: SpeI, SacI, BstXI, PmeI, NheI,MluI, HindIII, XmaI, SmaI, SalI. To 3′ end: SpeI, SacI, Sse83871, XhoI.The Soy FAD3-1A intron #5 PCR product is cloned into PCR2.1, resultingin KAWHIT03.0065.

[0176] Step 2—The soybean FAD3-1A intron #5 PCR product is then clonedinto an empty AMP vector by digesting KAWHIT03.0065 (Soybean FAD3-1Aintron #5 in pCR2.1) with SpeI and then the ends are filled in using theKlenow fragment of T4 Polymerase. pMON68526 (empty AMP vector) isdigested with HindIII and then the ends are filled in using the Klenowfragment of T4 Polymerase. The soybean FAD3-1A PCR product with therestriction sites described above is blunt-end ligated into pMON68526,resulting in pMON68541 (FAD3-1A PCR product in empty AMP vector).

[0177] Step 3—The soybean FAD 2-1A intron #1 is PCR amplified usingsoybean genomic DNA as template, with the following primers:

[0178] 5′ primer=18663=GGGCCCGGTAAATTAAATTGTGC (Adding Bsp120I site to5′ end);

[0179] 3′ primer=18664=CTGTGTCAAAGTATAAACAAGTTCAG. The resulting PCRproduct is cloned into PCR 2.1 creating KAWHIT03.0038.

[0180] Step 4—Soybean FAD 2-1A intron #1 PCR product in KAWHIT03.0038 iscloned into KAWHIT03.0032 (empty CM resistant vector with a multiplecloning site) using the restriction sites Bsp120I and EcoRI. Theresulting plasmid is KAWHIT03.0039 (Soybean FAD 2-1A intron #1 in emptyCM resistant vector).

[0181] Step 5—KAWHIT03.0039 is digested with AscI and HindIII andpMON68541 (FAD3-1A PCR product in empty AMP vector) is digested withMluI and HindIII. The Soybean FAD 2-1A intron #1 is then directionallycloned into pMON68541 to generate KAWHIT03.0071 (soybean FAD2-1A intron#1 with soybean FAD3-1A Intron #5).

[0182] Step 6-5′ and 3′ end portions of soybean FAD3-1A intron #4 arePCR amplified to create a 376 bp fragment using genomic DNA as templateand the following primers:

[0183] 5′ Primer of 5′ end=19034=GGGCCCAAATAGTGGGTGAAC (This primeradded a Bsp120I site to 5′ end)

[0184] 3′ Primer of 5′ end=18993=GAACTAAGGGACACAAC

[0185] 5′ Primer of 3′ end=18990=CTTAGTTCGCTCTTACCTGTGATC

[0186] 3′ Primer of 3′ end=18996=GTCCATTTTAGTACACCAC

[0187] The resulting PCR product is cloned into PCR 2.1 to formKAWHITO3.0067 containing the 5′ and 3′ ends of intron #4 from thesoybean FAD3-1A.

[0188] Step 7—KAWHIT03.0067 is cloned into KAWHIT03.0032 (empty CMresistant vector with a multiple cloning site) using the restrictionsites Bsp120I and EcoRI, resulting in plasmid KAWHIT03.0068.

[0189] Step 8—KAWHIT03.0068 (5′ and 3′ ends of intron #4 from thesoybean FAD3-1A in CM resistant Vector) is digested with AscI andHindIII and KAWHIT03.0071 (Soybean FAD2-1A intron #1 with soybeanFAD3-1A intron #5) is digested with MluI and HindIII. The 5′ and 3′ endsof intron #4 from the soybean FAD3-1A are directionally ligated intoKAWHIT03.0071 creating KAWHIT03.0075 (soybean FAD2-1A intron#1, soybeanFAD3-1A intron #4 ends and soybean FAD3-1A intron #5).

[0190] Step 9—5′ and 3′ end portions of soybean FATB intron #2 are PCRamplified to create a 374 bp fragment using genomic DNA as template andthe following primers:

[0191] 5′ Primer of 5′ end=19205=GGGCCCTTCTCGATTCTTTTCTC (Adding Bsp120Isite to 5′ end)

[0192] 3′ Primer of 5′ end=19147=CAGACAAGGCAAAGAAACAAGGGAG

[0193] 5′ Primer of 3′ end=19088=GCCTTGTCTGGTCCGATTGATTTCTCG

[0194] 3′ Primer of 3′ end=19089=CATGCATGCAAAATATACGCAAGTTAG Theresulting PCR product is cloned into PCR 2.1 to form KAWHIT03.0069.

[0195] Step 10—KAWHIT03.0069 (containing the 5′ and 3′ ends of Intron #2from the soybean FATB) is cloned into KAWHIT03.0032 (empty CM resistantvector with a multiple cloning site) using the restriction sites Bsp1201and EcoRI to create KAWHIT03.0070. (5′ and 3′ ends of intron #2 from thesoybean FATB in CM resistant vector).

[0196] Step 11—KAWHIT03.0070 (5′ and 3′ ends of intron #2 from thesoybean FATB in CM resistant vector) is digested with AscI and HindIIIand KAWHIT03.0075 (Soybean FAD2-1A intron #1, soybean FAD3-1A intron #4ends and soybean FAD3-1A intron #5) is digested with MluI and HindIII.The 5′ and 3′ ends of intron #2 from the soybean FATB are directionallyligated into KAWHIT03.0075 to generate KAWHIT03.0077 (Soybean FAD2-1Aintron #1, soybean FAD3-1A intron #4 ends, soybean FATB intron #2 endsand soybean FAD3-1A intron #5).

[0197] Step 12—Soybean FAD3-1B intron #4 is PCR amplified using genomicDNA as template and the following primers:

[0198] 5′ Primer=19516=CCCAAGCTTGGGGTATCCCATTTAACAC (Adding HindIII siteto 5′ end)

[0199] 3′ Primer=19515 GACCCGGGTCCTGTGAAATTACATATAGAC (Adding XmaCI siteto 3′ end)

[0200] The resulting PCR product is cloned into PCR 2.1 to formKAWHIT03.0090.

[0201] Step 13—To add the soybean FAD3-1B intron #4 into KAWHIT03.0077,plasmids KAWHIT03.0090 and KAWHIT03.0077 are digested with HindIII andXmaCI and directionally ligated to make KAWHIT03.0091 (Soybean FAD2-1Aintron#1, soybean FAD3-1A intron #4 ends, soybean FATB intron #2 ends,soybean FAD3-1A intron #4 and soybean FAD3-1A intron #5).

[0202] Step 14—KAWHIT03.0091 is digested with BstXI and SalI and thefragment containing the four introns (Soybean FAD2-1A intron #1, soybeanFAD3-1A intron #4 ends, soybean FATB intron #2 ends, soybean FAD3-1Aintron #4) is gel purified. In a different tube KAWHIT03.0091, is alsodigested with XhoI and Sse83871. The four intron fragment is then clonedback into KAWHIT03.0091 in the opposite orientation on the other site ofSoy FAD3-1A intron #5 to create KAWHIT03.0092 (soybean FAD2-1A intron #1sense, soybean FAD3-1A intron #4 ends sense, soybean FATB intron #2 endssense, soybean FAD3-1A intron #4 sense, spliceable soybean FAD3-1Aintron #5, soy FAD3-1B intron #4 anti-sense, soybean FATB intron #2 endsanti-sense, soybean FAD3-1A intron #4 ends anti-sense, soybean FAD2-1Aintron #1 anti-sense).

[0203] Step 15—To link the RNAi construct to the 7S alpha′ promoter andthe TML 3′, KAWHIT03.0092 and pMON68527 (7Sa′/TML3′ cassette) aredigested with SacI and ligated together to make KAWHIT03.0093 0092 (7Salpha′ promoter—FAD2-1A intron #1 sense, soybean FAD3-1A intron #4 endssense, soybean FATB intron #2 ends sense, soybean FAD3-1A intron #4sense, spliceable soybean FAD3-1A Intron #5, soy FAD3-1B intron #4anti-sense, soybean FATB intron #2 ends anti-sense, soybean FAD3-1Aintron #4 ends anti-sense, soybean FAD2-1A intron #1 anti-sense—TML3′).

[0204] Step 16—To introduce the assembled RNAi construct into pMON80612,which contains the selectable maker CP4 fused to the FMV promoter andthe RBCS 3′, KAWHIT03.0093 and pMON80612 are digested with NotI andligated together to form pMON68456 (illustrated in FIG. 4) comprising a7S alpha′ promoter operably linked to the intron series,double-stranded-RNA-forming construct of FAD2-1A intron #1 sense,soybean FAD3-1A intron #4 ends sense, soybean FATB intron #2 ends sense,soybean FAD3-1A intron #4 sense, spliceable soybean FAD3-1A intron #5,soy FAD3-1B intron #4 anti-sense, soybean FATB intron #2 endsanti-sense, soybean FAD3-1A intron #4 ends anti-sense, soybean FAD2-1Aintron #1 anti-sense and TML3′ terminator).

[0205] Representative sequences for FAD2-1A, FAD2-1B, FAD2-2B, FAD3-1A,FAD3-1B, and FAD3-1C introns include, without limitation, those setforth in U.S. application Ser. No. 10/176,149, filed Jun. 21, 2002, andU.S. patent application Ser. No. 09/638,508, filed Aug. 11, 2000, andU.S. Provisional Application Serial No. 60/151,224, filed Aug. 26, 1999,and U.S. Provisional Application Serial No. 60/172,128, filed Dec. 17,1999, all of which applications are herein incorporated by reference intheir entireties including, without limitation, their accompanyingsequence listings.

[0206] Representative sequences for FATB introns include, withoutlimitation, those set forth in U.S. Provisional Application Serial No.60/390,185, filed Jun. 21, 2002, which application is hereinincorporated by reference in its entirety, including without limitationits sequence listing.

Example 5 This Example Illustrates the Preparation of a Variety ofIntron dsRNA-Forming Constructs Which Can Suppress One or a Plurality ofGenes in Soybean

[0207] Using the step-wise method illustrated in Example 4, introndsRNA-forming vectors are constructed to have the following elements:

[0208] (1) 7S promoter—FAD2-1A sense intron—FAD3-1C sense intron—FAD3-1Asense intron—FAD3-1B sense intron—spliceable FAD3 intron #5—FAD3-1Banti-sense intron—FAD3-1A anti-sense intron—FAD3-1C anti-senseintron—FAD2-1A anti-sense intron—pea rbcS;

[0209] (2) 7S promoter—FAD2-1A sense intron—FAD3-1A sense intron—FAD3-1Bsense intron—spliceable FAD3 intron #5—FAD3-1B anti-sense intron—FAD3-1Aanti-sense intron—FAD2-1A anti-sense intron—pea rbcS;

[0210] (3) 7S promoter—FAD2-1A sense intron—FAD3-1A senseintron—spliceable FAD3 intron #5—FAD3-1A anti-sense intron—FAD2-1Aanti-sense intron—pea rbcS;

[0211] (4) 7S promoter—FAD2-1A sense intron—spliceable FAD3 intron#5—FAD2-1A anti-sense intron—pea rbcS;

[0212] (5) 7S promoter—FAD3-1A sense intron—spliceable FAD3 intron#5—FAD3-1A anti-sense intron—pea rbcS;

[0213] (6) 7S promoter—FAD2-1A sense intron—FAD3-1A sense3′UTR—spliceable FAD3 intron #5—FAD3-1A anti-sense 3′UTR—FAD2-1Aanti-sense intron—pea rbcS; and

[0214] (7) 7S promoter—FAD2-1A sense intron—FAD3-1A sense 3′UTR—FAD3-1Bsense 3′UTR—spliceable FAD3 intron #5—FAD3-1B anti-sense 3′UTR—FAD3-1Aanti-sense 3′UTR—FAD2-1A anti-sense intron—pea rbcS;

[0215] (8) 7S promoter—FATB sense intron I—FATB sense intronII—spliceable FAD3 intron #5—FATB anti-sense intron II—FATB anti-senseintron I—pea rbcS;

[0216] (9) 7S promoter—FATB sense intron II—FATB sense intronI—spliceable FAD3 intron #5—FATB anti-sense intron I—FATB anti-senseintron II—pea rbcS;

[0217] (10) 7S promoter—FATB sense intron—spliceable FAD3 intron #5—FATBanti-sense intron—pea rbcS;

[0218] (11) 7S promoter—FAD2-1A sense intron—FAD3-1C senseintron—FAD3-1A sense intron—FAD3-1B sense intron—FATB senseintron—spliceable FAD3 intron #5—FATB anti-sense intron—FAD3-1Banti-sense intron—FAD3-1A anti-sense intron—FAD3-1C anti-senseintron—FAD2-1A anti-sense intron—pea rbcS;

[0219] (12) 7S promoter—FAD2-1A sense intron—FAD3-1A senseintron—FAD3-1B sense intron—FATB sense intron—spliceable FAD3 intron#5—FATB anti-sense intron—FAD3-1B anti-sense intron—FAD3-1A anti-senseintron—FAD2-1A anti-sense intron—pea rbcS; and

[0220] (13) 7S promoter—FAD2-1A sense intron sense intron—FAD3-1A senseintron—FATB sense intron—spliceable FAD3 intron #5—FATB anti-senseintron—FAD3-1A anti-sense intron—FAD2-1A anti-sense intron—pea rbcS.

Example 6 This Example Illustrates Plant Transformation with theConstructs of this Invention to Produce Soybean Plants with SuppressedGenes

[0221] A transformation vector pMON68456 as prepared in Example 4 isused to introduce an intron double-stranded RNA-forming construct intosoybean for suppressing the A12 desaturase, A 15 desaturase, and FA TBgenes. The vector is stably introduced into soybean (Asgrow varietyA4922) via Agrobacterium tumefaciens strain ABI (Martinell, U.S. Pat.No. 6,384,301). The CP4 selectable marker allows transformed soybeanplants to be identified by selection on media containing glyphosateherbicide.

[0222] Fatty acid compositions are analyzed from seed of soybean linestransformed with the intron expression constructs using gaschromatography. R₁ pooled seed and R₁ single seed oil compositionsdemonstrate that the mono- and polyunsaturated fatty acid compositionswere altered in the oil of seeds from transgenic soybean lines ascompared to that of the seed from non-transformed soybean. For instance,FAD2 suppression provides plants with increased amount of oleic acidester compounds; FAD3 suppression provides plants with decreasedlinolenic acid ester compounds; and FATB suppression provides plantswith reduced saturated fatty ester compounds, e.g. palmitates andstearates. Selections can be made from such lines depending on thedesired relative fatty acid composition. Fatty acid compositions areanalyzed from seed of soybean lines transformed with constructs usinggas chromatography.

Example 7 This Example Illustrates Transient Expression Of Constructsfor Intron Double-Stranded RNA Gene Suppression

[0223] DNA containing the expression constructs for sense, antisense,and dsRNA expression of the Δ12 desaturase, Δ15 desaturase, and FATBintrons is transferred into the nucleus or the cytoplasm of tobaccomesophyll protoplasts. The DNA constructs illustrated in Examples 3, 4,5 and are introduced by microinjection as described (Crossway et al.,(1986) Mol. Gen. Genet. 202: 179-185). Transient gene suppression isobserved, e.g., by measuring RNA or fatty acid compound compositions.

[0224] All publications and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication or patent application was specificallyand individually indicated to be incorporated by reference.

1 55 1 420 DNA Glycine max 1 gtaaattaaa ttgtgcctgc acctcgggat atttcatgtggggttcatca tatttgttga 60 ggaaaagaaa ctcccgaaat tgaattatgc atttatatatcctttttcat ttctagattt 120 cctgaaggct taggtgtagg cacctagcta gtagctacaatatcagcact tctctctatt 180 gataaacaat tggctgtaat gccgcagtag aggacgatcacaacatttcg tgctggttac 240 tttttgtttt atggtcatga tttcactctc tctaatctctccattcattt tgtagttgtc 300 attatcttta gatttttcac tacctggttt aaaattgagggattgtagtt ctgttggtac 360 atattacaca ttcagcaaaa caactgaaac tcaactgaacttgtttatac tttgacacag 420 2 405 DNA Glycine max 2 gtatgatgct aaattaaattgtgcctgcac cccaggatat ttcatgtggg attcatcatt 60 tattgaggaa aactctccaaattgaatcgt gcatttatat tttttttcca tttctagatt 120 tcttgaaggc ttatggtataggcacctaca attatcagca cttctctcta ttgataaaca 180 attggctgta ataccacagtagagaacgat cacaacattt tgtgctggtt accttttgtt 240 ttatggtcat gatttcactctctctaatct gtcacttccc tccattcatt ttgtacttct 300 catatttttc acttcctggttgaaaattgt agttctcttg gtacatacta gtattagaca 360 ttcagcaaca acaactgaactgaacttctt tatactttga cacag 405 3 6220 DNA Glycine max 3 agcttggtaccgagctcgga tccactagta acggccgcca gtgtgctgga attcggcttc 60 tctctcaccctcctcttcac acattttctg tgcgctctaa caaacattct cgttcacact 120 ttcaggtacttttctctcct tatctcttta tctttattct ttcctacttt attgcttaaa 180 ccaatgctatctatgcttcg atctcgcctt cttattttcc acttcccttt tctcgcttga 240 tctaaccgttttcgccctcc gcgcttcgat tgactgagta catctacgat tctctgttct 300 ttcatttcatagatttcgtc tgattttggc taacttggtt tctgttgcgg ccgattctta 360 catatactgattgtttagca taaatgaact tgcttgttta gcactatctg catattttcg 420 tcacgcatctctttcggatc taaggatgaa tctcctattt cctccgtatt atttctcgta 480 tctcttgttctgtgctaatg ctccagaaaa tggcagcatt gtcttcttct ttgctgtata 540 agtgtttgtgttgtgaatct ggaagcgatt ttgcgtgagg taacttgcga cttcaactat 600 tatctttcagatctcgttaa tttattagct gctattaatt tgtgtgtgca gtgtcaaact 660 gaagcacacgactgcttaga agttagaatt tgactgactg ttcctctttg atttttttct 720 ttcttttctttgctwactcg gcctatttaa tgatctttat aaatagatta gtggaccact 780 tggttagttggtgagttatg aatattcgaa ttttctacca caagttgggt taaaaaaatc 840 tctgcaactacacgaggatt ttttatttta tttagaggaa actattctgt catccttttt 900 ccgattacacttttctatca gttgttttga aatatacacc ttaggaatat aatattaccc 960 ctttcggtcttaatataaat atattttaat tatttatatt ttatttaatg aaattatttt 1020 taaaatactttcatttaata gaatttttaa taaagttaaa gacttttatt gtgtagagtt 1080 taacgaagttaattagtttt cttagtaaat gtaaaatatg ccttttttgt tgtttataat 1140 ggagattggaaaaaatatac tttaattttt ttcaagtgat gaataattat ggatgttttg 1200 tcaatatttttgtcttgcta tacaactttc agtcttgcca ttaaataatt ttgaatgtgt 1260 tattgatatctctgaacaat atttagagac gaacataaat tttatatatt ttatataatt 1320 tctttttattacccttttat tatcaatttt gaaatttggt taatatctgt gtttcatttt 1380 gaggtctcaaatttgatata aggaggttca aaatgcgttg ctagccattt taaagattag 1440 caggagaggaaatgtttctg gacttaaatt taaaatatgc ttatttgttt ttcaagagag 1500 agagatcaatatttatataa tacacttgaa ttaatataca ccattgttgc aaaaaaaaaa 1560 aaatattagttgattgtgtg acaatatttt atattaaata taattagtta atttagttca 1620 agttgagttacatttttaca taccattctt agccgccact tttttatatt tatttgtagg 1680 aataacttttcatctgtatc aattttcccc gtctaataaa aagggtttga ctttttctta 1740 taatagagtttttttttttt tgctttaagt tattgtaaaa taattatttt attttttttg 1800 cctttgtaaattatgtatat ttaatgtttt aataggaaaa aaatgttatc aaaagcacta 1860 aaagactaaaattaaacaac cataatttgc aaagatgaaa ataaaaaaat aattttgtaa 1920 agataaaaaatgaaataaaa tagttaaatt ataggaattt aaaagctatt taaatcaaca 1980 aaagttaaagtttctgtaaa aaaagttcaa tttttttttt tattattgaa aaagttaaag 2040 ctaatgagcgttcgatttgg gttagtatgt agtatttatt attttcaaga ttttggattt 2100 tattgtcgatgtttctgatt tgaatataat tattttccat tcaacttgtg attttataag 2160 aaaaaaaaaggtacagaaaa aatcaagcgc tttttttatt tcaattagtg gaggtttcac 2220 tgaaatgggtaaagaatcta ttttgcaatc acaattatta ccggtattca actgcaacaa 2280 ggaacaaaattcctttcgta aatatacgga gaggaatcta ttttgacttg ttgaatttat 2340 ggtaaagtagaatttagaat ttaattatga gttgaagtaa ttttgaataa tttatatgtt 2400 aaatataaaattttgtacta agttttattc ataactttga ttctataata caaacataca 2460 taagttcaaaaataatttta attaaaatta attttatcaa tttttattca aacacgagtc 2520 taatttgcttgatgaattaa gaaaataagg aagaaaatat taaaaactag gagagaagtt 2580 aaagagaatttcatctttat tattctcagt tgtttcaaaa ataatgaaag gatagctata 2640 taatactgtaactgagccaa gaacatattt gccgtccgag taaccttttc ttttcttgtt 2700 ccgttttctccgccgatgaa gagagggaag ggaatgtatc tttgtattta tgttttcaaa 2760 gagttcgtgcataaaattgg tttaatcaaa tttttcataa gattattatt ttatgatttt 2820 ttaaaataaattagtaacta tattccgtaa gtcgtacaca gttatatgta gtaagtaaat 2880 tatattttaataattattat cttaaaattt tcttaagaac ttggttaaaa tatttttgtt 2940 tgaaaaagtttatgataact tttttttgtt gaaaaaaagt ttacgattat ctaactcgta 3000 cttagattatttctaattgg gatttattga agggtttttt aagtaaagaa attgtttctt 3060 atggtttcttttttattgga caaatttacg tagcaaagag tgtttcttaa aaacaagaca 3120 tgtatcctttgaaaaaaaac tatttctttg aaataaaaaa taatatttat ctggcacata 3180 ataatgttaaaattaaatca taattaggta aaaataaaat aaatataaaa gtatgagttt 3240 gttaagttttttataatttt ttattattaa agtaaaatta tgtatgattt ttttataatg 3300 atatgatattttagggatca caaaaaataa tgtggtgaat acaaaagtaa ctcaaaaaat 3360 tcatttagtaaattttcatt ggagatgcta ttattatgct ttctgattgc tttgtccaaa 3420 aaataaagaatgttttttta tttgaaaatt gaaaatttct gggtcatgtt aagatcttgt 3480 agacggtaacgtcggcctaa agttgtgtga ggggtgttgc atgcaccgat cattaattac 3540 tcgatatggaaaacgactga aataatttaa tttgatgttg ctaatattgg ccatccctct 3600 catcattattgtttttttat ttgtaacatg acatattctt gtgggtccgc tacggattgg 3660 gtgtttgttgccaaaaaata caaaatatct gtggaacaag gataaacagt cttgtttgtt 3720 taattgattgattgatgagt ttgcaagcta tatttttaat ttattttaat taaacttttg 3780 tgttttagttctacaatttt attcatcttg attttttttt tacttggcaa aatcatgatt 3840 ttttaatttttacttatgtt gaaaacaaat ttattgctaa aaaaacattt attctttttt 3900 tagagaaaaaacaaatttgt gatatgtagt gaatcaaatg aaaattttaa acataatata 3960 gaatactctacaaatcaatt ttgagtttct ttatcatttt atttatttat tgacatactt 4020 ctactttctgcaaagaccct gactcgtgga agatataggg aaggttatgg aagttagtgt 4080 attgtcatatctagctatct ttgctaattg aaaaagcctt ccctttgttt acagatctgg 4140 ataaggttgcatgtttattc ttttcaactg tgaatggttc tttgcatctt ttttagtata 4200 tgagattaatgttttaatta ggaagaagct tttagaacat cacccgaatc caattcgttt 4260 tggtttctgtgatcttgatg taaatctata ctaatttggt ttgggcagaa gaaaatgttc 4320 tttgctcaagtcctctagga cgaaaatata aatataacag ggtatatcag atctctattc 4380 ttctgtgggtaatgatagca tgtttctgtt gttttcttat tcttcattgg tcatgataac 4440 ctgctaattctatttgccac gattgagatg aaaaggtaat gaactagtaa acaataatga 4500 gaagaatatgtcgctactat tgttgaaacg gttacgccag gcacttgagt atgatgcact 4560 attttaattaatgcattttt tttgctttga tgagaacgca cattgttcat tctgattcgg 4620 tgagtttagaaactattgct gataatcctt gatttaagat tttagtcttg ttcatgttca 4680 ttaaaagtgttgtaaaaaaa tgcactgata tgtcatgtgc agattgtgtg aagatggggg 4740 cgggtggccgaactgatgtt cctcctgcca acaggaagtc agaggttgac cctttgaagc 4800 gggtgccatttgaaaaacct ccatttagtc tcagccaaat caagaaggtc attccacctc 4860 actgtttccagcgttctgtt ttccgctcat tctcctatgt tgtttacgac ctcaccatag 4920 ccttctgcctctattatgtt gccacccatt acttccacct ccttcccagc cctctctctt 4980 tcttggcatggccaatctac tgggctgtcc aaggttgcat ccttactgga gtttgggtca 5040 ttgcccatgagtgtggccac catgcattca gtgactacca gttgcttgat gatattgttg 5100 gccttgtcctccactccggt ctcctagtcc catacttttc atggaaatac agccatcgcc 5160 gtcaccactccaacactggt tctcttgagc gggatgaagt atttgtgcca aagcagaagt 5220 cctgtatcaagtggtactct aaatacctta acaatcctcc aggcagagtc ctcactcttg 5280 ctgtcaccctcacacttggt tggcccttgt acttggcttt aaatgtttct ggaaggcctt 5340 atgatagatttgcttgccac tatgacccat atggtcccat ttactctgat cgtgaacgac 5400 ttcaaatatatatatcagat gcaggagtac ttgcagtatg ctatggcctt ttccgtcttg 5460 ccatggcaaaaggacttgcc tgggtggtgt gtgtttatgg agttccattg ctagtggtca 5520 atggatttttggtgttgatt acattcttgc agcatactca ccctgcattg ccacattaca 5580 cttcctctgagtgggactgg ttgagaggag ctttagcaac agtggataga gattatggaa 5640 tcctgaacaaggtcttccat aatattacag acactcatgt agcacatcac ttgttctcca 5700 caatgccacattatcatgca atggaggcta caaaggcaat aaaacccatt ttgggagagt 5760 attatcggtttgatgagact ccatttgtca aggcaatgtg gagagaggca agagagtgta 5820 tttatgtggagccagatcaa agtaccgaga gcaaaggtgt attttggtac aacaataagt 5880 tgtgatgattaatgtagccg aggcttcttt gaactttccc ttgtgactgt ttagtatcat 5940 ggttgcttattgggaataat tttgttgaac cctgatgttg gtagtaagta tctagacagt 6000 tgcatagcggttttgtttac agaataagat atagcctctc tgaacagttt gattattgca 6060 ccatggtttgcaatcggtgc atgtcgacca agtttctcaa gactgtggag aagcttattc 6120 ttgttccagttcttgaatcc aagttgttac cgtattctgt aagccgaatt ctgcagatat 6180 ccatcacactggcggccgct cgagcatgca tctagagggc 6220 4 4597 DNA Glycine max 4gtacttttct ctccttatct ctttatcttt attctttcct actttattgc ttaaaccaat 60gctatctatg cttcgatctc gccttcttat tttccacttc ccttttctcg cttgatctaa 120ccgttttcgc cctccgcgct tcgattgact gagtacatct acgattctct gttctttcat 180ttcatagatt tcgtctgatt ttggctaact tggtttctgt tgcggccgat tcttacatat 240actgattgtt tagcataaat gaacttgctt gtttagcact atctgcatat tttcgtcacg 300catctctttc ggatctaagg atgaatctcc tatttcctcc gtattatttc tcgtatctct 360tgttctgtgc taatgctcca gaaaatggca gcattgtctt cttctttgct gtataagtgt 420ttgtgttgtg aatctggaag cgattttgcg tgaggtaact tgcgacttca actattatct 480ttcagatctc gttaatttat tagctgctat taatttgtgt gtgcagtgtc aaactgaagc 540acacgactgc ttagaagtta gaatttgact gactgttcct ctttgatttt tttctttctt 600ttctttgctw actcggccta tttaatgatc tttataaata gattagtgga ccacttggtt 660agttggtgag ttatgaatat tcgaattttc taccacaagt tgggttaaaa aaatctctgc 720aactacacga ggatttttta ttttatttag aggaaactat tctgtcatcc tttttccgat 780tacacttttc tatcagttgt tttgaaatat acaccttagg aatataatat tacccctttc 840ggtcttaata taaatatatt ttaattattt atattttatt taatgaaatt atttttaaaa 900tactttcatt taatagaatt tttaataaag ttaaagactt ttattgtgta gagtttaacg 960aagttaatta gttttcttag taaatgtaaa atatgccttt tttgttgttt ataatggaga 1020ttggaaaaaa tatactttaa tttttttcaa gtgatgaata attatggatg ttttgtcaat 1080atttttgtct tgctatacaa ctttcagtct tgccattaaa taattttgaa tgtgttattg 1140atatctctga acaatattta gagacgaaca taaattttat atattttata taatttcttt 1200ttattaccct tttattatca attttgaaat ttggttaata tctgtgtttc attttgaggt 1260ctcaaatttg atataaggag gttcaaaatg cgttgctagc cattttaaag attagcagga 1320gaggaaatgt ttctggactt aaatttaaaa tatgcttatt tgtttttcaa gagagagaga 1380tcaatattta tataatacac ttgaattaat atacaccatt gttgcaaaaa aaaaaaaata 1440ttagttgatt gtgtgacaat attttatatt aaatataatt agttaattta gttcaagttg 1500agttacattt ttacatacca ttcttagccg ccactttttt atatttattt gtaggaataa 1560cttttcatct gtatcaattt tccccgtcta ataaaaaggg tttgactttt tcttataata 1620gagttttttt ttttttgctt taagttattg taaaataatt attttatttt ttttgccttt 1680gtaaattatg tatatttaat gttttaatag gaaaaaaatg ttatcaaaag cactaaaaga 1740ctaaaattaa acaaccataa tttgcaaaga tgaaaataaa aaaataattt tgtaaagata 1800aaaaatgaaa taaaatagtt aaattatagg aatttaaaag ctatttaaat caacaaaagt 1860taaagtttct gtaaaaaaag ttcaattttt ttttttatta ttgaaaaagt taaagctaat 1920gagcgttcga tttgggttag tatgtagtat ttattatttt caagattttg gattttattg 1980tcgatgtttc tgatttgaat ataattattt tccattcaac ttgtgatttt ataagaaaaa 2040aaaaggtaca gaaaaaatca agcgcttttt ttatttcaat tagtggaggt ttcactgaaa 2100tgggtaaaga atctattttg caatcacaat tattaccggt attcaactgc aacaaggaac 2160aaaattcctt tcgtaaatat acggagagga atctattttg acttgttgaa tttatggtaa 2220agtagaattt agaatttaat tatgagttga agtaattttg aataatttat atgttaaata 2280taaaattttg tactaagttt tattcataac tttgattcta taatacaaac atacataagt 2340tcaaaaataa ttttaattaa aattaatttt atcaattttt attcaaacac gagtctaatt 2400tgcttgatga attaagaaaa taaggaagaa aatattaaaa actaggagag aagttaaaga 2460gaatttcatc tttattattc tcagttgttt caaaaataat gaaaggatag ctatataata 2520ctgtaactga gccaagaaca tatttgccgt ccgagtaacc ttttcttttc ttgttccgtt 2580ttctccgccg atgaagagag ggaagggaat gtatctttgt atttatgttt tcaaagagtt 2640cgtgcataaa attggtttaa tcaaattttt cataagatta ttattttatg attttttaaa 2700ataaattagt aactatattc cgtaagtcgt acacagttat atgtagtaag taaattatat 2760tttaataatt attatcttaa aattttctta agaacttggt taaaatattt ttgtttgaaa 2820aagtttatga taactttttt ttgttgaaaa aaagtttacg attatctaac tcgtacttag 2880attatttcta attgggattt attgaagggt tttttaagta aagaaattgt ttcttatggt 2940ttctttttta ttggacaaat ttacgtagca aagagtgttt cttaaaaaca agacatgtat 3000cctttgaaaa aaaactattt ctttgaaata aaaaataata tttatctggc acataataat 3060gttaaaatta aatcataatt aggtaaaaat aaaataaata taaaagtatg agtttgttaa 3120gttttttata attttttatt attaaagtaa aattatgtat gattttttta taatgatatg 3180atattttagg gatcacaaaa aataatgtgg tgaatacaaa agtaactcaa aaaattcatt 3240tagtaaattt tcattggaga tgctattatt atgctttctg attgctttgt ccaaaaaata 3300aagaatgttt ttttatttga aaattgaaaa tttctgggtc atgttaagat cttgtagacg 3360gtaacgtcgg cctaaagttg tgtgaggggt gttgcatgca ccgatcatta attactcgat 3420atggaaaacg actgaaataa tttaatttga tgttgctaat attggccatc cctctcatca 3480ttattgtttt tttatttgta acatgacata ttcttgtggg tccgctacgg attgggtgtt 3540tgttgccaaa aaatacaaaa tatctgtgga acaaggataa acagtcttgt ttgtttaatt 3600gattgattga tgagtttgca agctatattt ttaatttatt ttaattaaac ttttgtgttt 3660tagttctaca attttattca tcttgatttt ttttttactt ggcaaaatca tgatttttta 3720atttttactt atgttgaaaa caaatttatt gctaaaaaaa catttattct ttttttagag 3780aaaaaacaaa tttgtgatat gtagtgaatc aaatgaaaat tttaaacata atatagaata 3840ctctacaaat caattttgag tttctttatc attttattta tttattgaca tacttctact 3900ttctgcaaag accctgactc gtggaagata tagggaaggt tatggaagtt agtgtattgt 3960catatctagc tatctttgct aattgaaaaa gccttccctt tgtttacaga tctggataag 4020gttgcatgtt tattcttttc aactgtgaat ggttctttgc atctttttta gtatatgaga 4080ttaatgtttt aattaggaag aagcttttag aacatcaccc gaatccaatt cgttttggtt 4140tctgtgatct tgatgtaaat ctatactaat ttggtttggg cagaagaaaa tgttctttgc 4200tcaagtcctc taggacgaaa atataaatat aacagggtat atcagatctc tattcttctg 4260tgggtaatga tagcatgttt ctgttgtttt cttattcttc attggtcatg ataacctgct 4320aattctattt gccacgattg agatgaaaag gtaatgaact agtaaacaat aatgagaaga 4380atatgtcgct actattgttg aaacggttac gccaggcact tgagtatgat gcactatttt 4440aattaatgca ttttttttgc tttgatgaga acgcacattg ttcattctga ttcggtgagt 4500ttagaaacta ttgctgataa tccttgattt aagattttag tcttgttcat gttcattaaa 4560agtgttgtaa aaaaatgcac tgatatgtca tgtgcag 4597 5 191 DNA Glycine max 5gtaataattt ttgtgtttct tactcttttt tttttttttt tgtttatgat atgaatctca 60cacattgttc tgttatgtca tttcttcttc atttggcttt agacaactta aatttgagat 120ctttattatg tttttgctta tatggtaaag tgattcttca ttatttcatt cttcattgat 180tgaattgaac a 191 6 346 DNA Glycine max 6 ttagttcata ctggcttttttgtttgttca tttgtcattg aaaaaaaatc ttttgttgat 60 tcaattattt ttatagtgtgtttggaagcc cgtttgagaa aataagaaat cgcatctgga 120 atgtgaaagt tataactatttagcttcatc tgtcgttgca agttctttta ttggttaaat 180 ttttatagcg tgctaggaaacccattcgag aaaataagaa atcacatctg gaatgtgaaa 240 gttataactg ttagcttctgagtaaacgtg gaaaaaccac attttggatt tggaaccaaa 300 ttttatttga taaatgacaaccaaattgat tttgatggat tttgca 346 7 142 DNA Glycine max 7 gtatgtgattaattgcttct cctatagttg ttcttgattc aattacattt tatttatttg 60 gtaggtccaagaaaaaaggg aatctttatg cttcctgagg ctgttcttga acatggctct 120 tttttatgtgtcattatctt ag 142 8 1228 DNA Glycine max 8 taacaaaaat aaatagaaaatagtgggtga acacttaaat gcgagatagt aatacctaaa 60 aaaagaaaaa aatataggtataataaataa tataactttc aaaataaaaa gaaatcatag 120 agtctagcgt agtgtttggagtgaaatgat gttcacctac cattactcaa agattttgtt 180 gtgtccctta gttcattcttattattttac atatcttact tgaaaagact ttttaattat 240 tcattgagat cttaaagtgactgttaaatt aaaataaaaa acaagtttgt taaaacttca 300 aataaataag agtgaagggagtgtcatttg tcttctttct tttattgcgt tattaatcac 360 gtttctcttc tcttttttttttttcttctc tgctttccac ccattatcaa gttcatgtga 420 agcagtggcg gatctatgtaaatgagtggg gggcaattgc acccacaaga ttttattttt 480 tatttgtaca ggaataataaaataaaactt tgcccccata aaaaataaat attttttctt 540 aaaataatgc aaaataaatataagaaataa aaagagaata aattattatt aattttatta 600 ttttgtactt tttatttagtttttttagcg gttagatttt tttttcatga cattatgtaa 660 tcttttaaaa gcatgtaatatttttatttt gtgaaaataa atataaatga tcatattagt 720 ctcagaatgt ataaactaataataatttta tcactaaaag aaattctaat ttagtccata 780 aataagtaaa acaagtgacaattatatttt atatttactt aatgtgaaat aatacttgaa 840 cattataata aaacttaatgacaggagata ttacatagtg ccataaagat attttaaaaa 900 ataaaatcat taatacactgtactactata taatattcga tatatatttt taacatgatt 960 ctcaatagaa aaattgtattgattatattt tattagacat gaatttacaa gccccgtttt 1020 tcatttatag ctcttacctgtgatctattg ttttgcttcg ctgtttttgt tggtcaaggg 1080 acttagatgt cacaatattaatactagaag taaatattta tgaaaacatg taccttacct 1140 caacaaagaa agtgtggtaagtggcaacac acgtgttgca tttttggccc agcaataaca 1200 cgtgtttttg tggtgtactaaaatggac 1228 9 625 DNA Glycine max 9 gtacatttta ttgcttattc acctaaaaacaatacaatta gtacatttgt tttatctctt 60 ggaagttagt cattttcagt tgcatgattctaatgctctc tccattctta aatcatgttt 120 tcacacccac ttcatttaaa ataagaacgtgggtgttatt ttaatttcta ttcactaaca 180 tgagaaatta acttatttca agtaataattttaaaatatt tttatgctat tattttatta 240 caaataatta tgtatattaa gtttattgattttataataa ttatattaaa attatatcga 300 tattaatttt tgattcactg atagtgttttatattgttag tactgtgcat ttattttaaa 360 attggcataa ataatatatg taaccagctcactatactat actgggagct tggtggtgaa 420 aggggttccc aaccctcctt tctaggtgtacatgctttga tacttctggt accttcttat 480 atcaatataa attatatttt gctgataaaaaaacatggtt aaccattaaa ttcttttttt 540 aaaaaaaaaa ctgtatctaa actttgtattattaaaaaga agtctgagat taacaataaa 600 ctaacactca tttggattca ctgca 625 1098 DNA Glycine max 10 ggtgagtgat tttttgactt ggaagacaac aacacattattattataata tggttcaaaa 60 caatgacttt ttctttatga tgtgaactcc atttttta 98 11115 DNA Glycine max 11 ggtaactaaa ttactcctac attgttactt tttcctccttttttttatta tttcaattct 60 ccaattggaa atttgaaata gttaccataa ttatgtaattgtttgatcat gtgca 115 12 148 DNA Glycine max FAD3-1B intron 3c 12gtaatctcac tctcacactt tctttataca tcgcacgcca gtgtgggtta tttgcaacct 60acaccgaagt aatgccctat aattaatgag gttaacacat gtccaagtcc aatattttgt 120tcacttattt gaacttgaac atgtgtag 148 13 361 DNA Glycine max FAD3-1B intron4 13 gtatcccatt taacacaatt tgtttcatta acattttaag agaatttttt tttcaaaata60 gttttcgaaa ttaagcaaat accaagcaaa ttgttagatc tacgcttgta cttgttttaa 120agtcaaattc atgaccaaat tgtcctcaca agtccaaacc gtccactatt ttattttcac 180ctactttata gcccaatttg ccatttggtt acttcagaaa agagaacccc atttgtagta 240aatatattat ttatgaatta tggtagtttc aacataaaac atacttatgt gcagttttgc 300catccttcaa aagaaggtag aaacttactc catgttactc tgtctatatg taatttcaca 360 g361 14 1037 DNA Glycine max 14 gtaacaaaaa taaatagaaa atagtgagtgaacacttaaa tgttagatac taccttcttc 60 ttcttttttt tttttttttt gaggttaatgctagataata gctagaaaga gaaagaaaga 120 caaatatagg taaaaataaa taatataacctgggaagaag aaaacataaa aaaagaaata 180 atagagtcta cgtaatgttt ggatttttgagtgaaatggt gttcacctac cattactcaa 240 agattctgtt gtctacgtag tgtttggactttggagtgaa atggtgttca cctaccatta 300 ctcagattct gttgtgtccc ttagttactgtcttatattc ttagggtata ttctttattt 360 tacatccttt tcacatctta cttgaaaagattttaattat tcattgaaat attaacgtga 420 cagttaaatt aaaataataa aaaattcgttaaaacttcaa ataaataaga gtgaaaggat 480 catcattttt cttctttctt ttattgcgttattaatcatg cttctcttct tttttttctt 540 cgctttccac ccatatcaaa ttcatgtgaagtatgagaaa atcacgattc aatggaaagc 600 tacaggaacy ttttttgttt tgtttttataatcggaatta atttatactc cattttttca 660 caataaatgt tacttagtgc cttaaagataatatttgaaa aattaaaaaa attattaata 720 cactgtacta ctatataata tttgacatatatttaacatg attttctatt gaaaatttgt 780 atttattatt ttttaatcaa aacccataaggcattaattt acaagaccca tttttcattt 840 atagctttac ctgtgatcat ttatagctttaagggactta gatgttacaa tcttaattac 900 aagtaaatat ttatgaaaaa catgtgtcttaccccttaac cttacctcaa caaagaaagt 960 gtgataagtg gcaacacacg tgttgcttttttggcccagc aataacacgt gtttttgtgg 1020 tgtacaaaaa tggacag 1037 15 4497DNA Glycine max 15 cttgcttggt aacaacgtcg tcaagttatt attttgttcttttttttttt atcatatttc 60 ttattttgtt ccaagtatgt catattttga tccatcttgacaagtagatt gtcatgtagg 120 aataggaata tcactttaaa ttttaaagca ttgattagtctgtaggcaat attgtcttct 180 tcttcctcct tattaatatt ttttattctg ccttcaatcaccagttatgg gagatggatg 240 taatactaaa taccatagtt gttctgcttg aagtttagttgtatagttgt tctgcttgaa 300 gtttagttgt gtgtaatgtt tcagcgttgg cttcccctgtaactgctaca atggtactga 360 atatatattt tttgcattgt tcattttttt cttttacttaatcttcattg ctttgaaatt 420 aataaaacaa aaagaaggac cgaatagttt gaagtttgaactattgccta ttcatgtaac 480 ttattcaccc aatcttatat agtttttctg gtagagatcattttaaattg aaggatataa 540 attaagagga aatacttgta tgtgatgtgt ggcaatttggaagatcatgc gtagagagtt 600 taatggcagg ttttgcaaat tgacctgtag tcataattacactgggccct ctcggagttt 660 tgtgcctttt tgttgtcgct gtgtttggtt ctgcatgttagcctcacaca gatatttagt 720 agttgttgtt ctgcatataa gcctcacacg tatactaaacgagtgaacct caaaatcatg 780 gccttacacc tattgagtga aattaatgaa cagtgcatgtgagtatgtga ctgtgacaca 840 acccccggtt ttcatattgc aatgtgctac tgtggtgattaaccttgcta cactgtcgtc 900 cttgtttgtt tccttatgta tattgatacc ataaattattactagtatat cattttatat 960 tgtccatacc attacgtgtt tatagtctct ttatgacatgtaattgaatt ttttaattat 1020 aaaaaataat aaaacttaat tacgtactat aaagagatgctcttgactag aattgtgatc 1080 tcctagtttc ctaaccatat actaatattt gcttgtattgatagcccctc cgttcccaag 1140 agtataaaac tgcatcgaat aatacaagcc actaggcatggtaaattaaa ttgtgcctgc 1200 acctcgggat atttcatgtg gggttcatca tatttgttgaggaaaagaaa ctcccgaaat 1260 tgaattatgc atttatatat cctttttcat ttctagatttcctgaaggct taggtgtagg 1320 cacctagcta gtagctacaa tatcagcact tctctctattgataaacaat tggctgtaat 1380 gccgcagtag aggacgatca caacatttcg tgctggttactttttgtttt atggtcatga 1440 tttcactctc tctaatctct ccattcattt tgtagttgtcattatcttta gatttttcac 1500 tacctggttt aaaattgagg gattgtagtt ctgttggtacatattacaca ttcagcaaaa 1560 caactgaaac tcaactgaac ttgtttatac tttgacacagggtctagcaa aggaaacaac 1620 aatgggaggt agaggtcgtg tggcaaagtg gaagttcaagggaagaagcc tctctcaagg 1680 gttccaaaca caaagccacc attcactgtt ggccaactcaagaaagcaat tccaccacac 1740 tgctttcagc gctccctcct cacttcattc tcctatgttgtttatgacct ttcatttgcc 1800 ttcattttct acattgccac cacctacttc cacctccttcctcaaccctt ttccctcatt 1860 gcatggccaa tctattgggt tctccaaggt tgccttctcactggtgtgtg ggtgattgct 1920 cacgagtgtg gtcaccatgc cttcagcaag taccaatgggttgatgatgt tgtgggtttg 1980 acccttcact caacactttt agtcccttat ttctcatggaaaataagcca tcgccgccat 2040 cactccaaca caggttccct tgaccgtgat gaagtgtttgtcccaaaacc aaaatccaaa 2100 gttgcatggt tttccaagta cttaaacaac cctctaggaagggctgtttc tcttctcgtc 2160 acactcacaa tagggtggcc tatgtattta gccttcaatgtctctggtag accctatgat 2220 agttttgcaa gccactacca cccttatgct cccatatattctaaccgtga gaggcttctg 2280 atctatgtct ctgatgttgc tttgttttct gtgacttactctctctaccg tgttgcaacc 2340 ctgaaagggt tggtttggct gctatgtgtt tatggggtgcctttgctcat tgtgaacggt 2400 tttcttgtga ctatcacata tttgcagcac acacactttgccttgcctca ttacgattca 2460 tcagaatggg actggctgaa gggagctttg gcaactatggacagagatta tgggattctg 2520 aacaaggtgt ttcatcacat aactgatact catgtggctcaccatctctt ctctacaatg 2580 ccacattacc atgcaatgga ggcaaccaat gcaatcaagccaatattggg tgagtactac 2640 caatttgatg acacaccatt ttacaaggca ctgtggagagaagcgagaga gtgcctctat 2700 gtggagccag atgaaggaac atccgagaag ggcgtgtattggtacaggaa caagtattga 2760 tggagcaacc aatgggccat agtgggagtt atggaagttttgtcatgtat tagtacataa 2820 ttagtagaat gttataaata agtggatttg ccgcgtaatgactttgtgtg tattgtgaaa 2880 cagcttgttg cgatcatggt tataatgtaa aaataattctggtattaatt acatgtggaa 2940 agtgttctgc ttatagcttt ctgcctaaaa tgcacgctgcacgggacaat atcattggta 3000 atttttttaa aatctgaatt gaggctactc ataatactatccataggaca tcaaagacat 3060 gttgcattga ctttaagcag aggttcatct agaggattactgcataggct tgaactacaa 3120 gtaatttaag ggacgagagc aactttagct ctaccacgtcgttttacaag gttattaaaa 3180 tcaaattgat cttattaaaa ctgaaaattt gtaataaaatgctattgaaa aattaaaata 3240 tagcaaacac ctaaattgga ctgattttta gattcaaatttaataattaa tctaaattaa 3300 acttaaattt tataatatat gtcttgtaat atatcaagttttttttttta ttattgagtt 3360 tggaaacata taataaggaa cattagttaa tattgataatccactaagat cgacttagta 3420 ttacagtatt tggatgattt gtatgagata ttcaaacttcactcttatca taatagagac 3480 aaaagttaat actgatggtg gagaaaaaaa aatgttattgggagcatatg gtaagataag 3540 acggataaaa atatgctgca gcctggagag ctaatgtattttttggtgaa gttttcaagt 3600 gacaactatt catgatgaga acacaataat attttctacttacctatccc acataaaata 3660 ctgattttaa taatgatgat aaataatgat taaaatatttgattctttgt taagagaaat 3720 aaggaaaaca taaatattct catggaaaaa tcagcttgtaggagtagaaa ctttctgatt 3780 ataattttaa tcaagtttaa ttcattcttt taattttattattagtacaa aatcattctc 3840 ttgaatttag agatgtatgt tgtagcttaa tagtaattttttatttttat aataaaattc 3900 aagcagtcaa atttcatcca aataatcgtg ttcgtgggtgtaagtcagtt attccttctt 3960 atcttaatat acacgcaaag gaaaaaataa aaataaaattcgaggaagcg cagcagcagc 4020 tgataccacg ttggttgacg aaactgataa aaagcgctgtcattgtgtct ttgtttgatc 4080 atcttcacaa tcacatctcc agaacacaaa gaagagtgacccttcttctt gttattccac 4140 ttgcgttagg tttctacttt cttctctctc tctctctctctcttcattcc tcatttttcc 4200 ctcaaacaat caatcaattt tcattcagat tcgtaaatttctcgattaga tcacggggtt 4260 aggtctccca ctttatcttt tcccaagcct ttctctttccccctttccct gtctgcccca 4320 taaaattcag gatcggaaac gaactgggtt cttgaatttcactctagatt ttgacaaatt 4380 cgaagtgtgc atgcactgat gcgacccact cccccttttttgcattaaac aattatgaat 4440 tgaggttttt cttgcgatca tcattgcttg aattgaatcatattaggttt agattct 4497 16 18 DNA Artificial sequence PCR primer 16atacaagcca ctaggcat 18 17 26 DNA Artificial sequence PCR primer 17gattggccat gcaatgaggg aaaagg 26 18 778 DNA Artificial sequencemisc_feature (1)..(778) unsure at all n locations 18 atacaagccactaggcatgg taaattaaat tgtgcctgca cctcgggata tttcatgtgg 60 ggttcatcatatttgttgag gaaaagaaac tcccgaaatt gaattatgca tttatatatc 120 ctttttcatttctagatttc ctgaaggctt aggtgtaggc acctagctag tagctacaat 180 atcagcacttctctctattg ataaacaatt ggctgtaatg ccgcagtaga ggacgatcac 240 aacatttcgtgctggttact ttttgtttta tggtcatgat ttcactctct ctaatctctc 300 cattcattttgtagttgtca ttatctttag atttttcact acctggttta aaattgaggg 360 attgtagttctgttggtaca tattacacat tcagcaaaac aactgaaact caactgaact 420 tgtttatactttgacacagg gtctagcaaa ggaaacaaca atgggaggta gaggtcgtgt 480 ggccaaagtggaagttcaag ggaagaagcc tctctcaagg gttccaaaca caaagccacc 540 attcactgttggccaactca agaaagcaat tccaccacac tgctttcagc gctccctcct 600 cacttcattctcctatgttg tttatgacct ttcatttgcc ttcattttct acattgccac 660 cacctacttccacctccttc ctcaaccctt ttccctcatt gcatggccaa tcaagccgaa 720 ttctgcagatatccatcaca tggcggcggn tggngnaggn ntntanaggg cccaattc 778 19 2463 DNAGlycine max 19 actatagggc acgcgtggtc gacggcccgg gctggtcctc ggtgtgactcagccccaagt 60 gacgccaacc aaacgcgtcc taactaaggt gtagaagaaa cagatagtatataagtatac 120 catataagag gagagtgagt ggagaagcac ttctcctttt tttttctctgttgaaattga 180 aagtgttttc cgggaaataa ataaaataaa ttaaaatctt acacactctaggtaggtact 240 tctaatttaa tccacacttt gactctatat atgttttaaa aataattataatgcgtactt 300 acttcctcat tatactaaat ttaacatcga tgattttatt ttctgtttctcttctttcca 360 cctacataca tcccaaaatt tagggtgcaa ttttaagttt attaacacatgtttttagct 420 gcatgctgcc tttgtgtgtg ctcaccaaat tgcattcttc tctttatatgttgtatttga 480 attttcacac catatgtaaa caagattacg tacgtgtcca tgatcaaatacaaatgctgt 540 cttatactgg caatttgata aacagccgtc cattttttct ttttctctttaactatatat 600 gctctagaat ctctgaagat tcctctgcca tcgaatttct ttcttggtaacaacgtcgtc 660 gttatgttat tattttattc tatttttatt ttatcatata tatttcttattttgttcgaa 720 gtatgtcata ttttgatcgt gacaattaga ttgtcatgta ggagtaggaatatcacttta 780 aaacattgat tagtctgtag gcaatattgt cttctttttc ctcctttattaatatatttt 840 gtcgaagttt taccacaagg ttgattcgct ttttttgtcc ctttctcttgttctttttac 900 ctcaggtatt ttagtctttc atggattata agatcactga gaagtgtatgcatgtaatac 960 taagcaccat agctgttctg cttgaattta tttgtgtgta aattgtaatgtttcagcgtt 1020 ggctttccct gtagctgcta caatggtact gtatatctat tttttgcattgttttcattt 1080 tttcttttac ttaatcttca ttgctttgaa attaataaaa caatataatatagtttgaac 1140 tttgaactat tgcctattca tgtaattaac ttattcactg actcttattgtttttctggt 1200 agaattcatt ttaaattgaa ggataaatta agaggcaata cttgtaaattgacctgtcat 1260 aattacacag gaccctgttt tgtgcctttt tgtctctgtc tttggttttgcatgttagcc 1320 tcacacagat atttagtagt tgttctgcat acaagcctca cacgtatactaaaccagtgg 1380 acctcaaagt catggcctta cacctattgc atgcgagtct gtgacacaacccctggtttc 1440 catattgcaa tgtgctacgc cgtcgtcctt gtttgtttcc atatgtatattgataccatc 1500 aaattattat atcatttata tggtctggac cattacgtgt actctttatgacatgtaatt 1560 gagtttttta attaaaaaaa tcaatgaaat ttaactacgt agcatcatatagagataatt 1620 gactagaaat ttgatgactt attctttcct aatcatattt tcttgtattgatagccccgc 1680 tgtccctttt aaactcccga gagagtataa aactgcatcg aatattacaagatgcactct 1740 tgtcaaatga agggggggaa atgatactac aagccactag gcatggtatgatgctaaatt 1800 aaattgtgcc tgcaccccag gatatttcat gtgggattca tcatttattgaggaaaactc 1860 tccaaattga atcgtgcatt tatatttttt ttccatttct agatttcttgaaggcttatg 1920 gtataggcac ctacaattat cagcacttct ctctattgat aaacaattggctgtaatacc 1980 acagtagaga acgatcacaa cattttgtgc tggttacctt ttgttttatggtcatgattt 2040 cactctctct aatctgtcac ttccctccat tcattttgta cttctcatatttttcacttc 2100 ctggttgaaa attgtagttc tcttggtaca tactagtatt agacattcagcaacaacaac 2160 tgaactgaac ttctttatac tttgacacag ggtctagcaa aggaaacaataatgggaggt 2220 ggaggccgtg tggccaaagt tgaaattcag cagaagaagc ctctctcaagggttccaaac 2280 acaaagccac cattcactgt tggccaactc aagaaagcca ttccaccgcactgctttcag 2340 cgttccctcc tcacttcatt gtcctatgtt gtttatgacc tttcattggctttcattttc 2400 tacattgcca ccacctactt ccacctcctc cctcacccct tttccctcattgcatggcca 2460 atc 2463 20 44 DNA Artificial sequence PCR primer 20cuacuacuac uactcgagac aaagccttta gcctttagcc tatg 44 21 36 DNA Artificialsequence PCR primer 21 caucaucauc auggatccca tgtctctcta tgcaag 36 221704 DNA Glycine max 22 actatagggc acgcgtggtc gacggcccgg gctggtcctcggtgtgactc agccccaagt 60 gacgccaacc aaacgcgtcc taactaaggt gtagaagaaacagatagtat ataagtatac 120 catataagag gagagtgagt ggagaagcac ttctcctttttttttctctg ttgaaattga 180 aagtgttttc cgggaaataa ataaaataaa ttaaaatcttacacactcta ggtaggtact 240 tctaatttaa tccacacttt gactctatat atgttttaaaaataattata atgcgtactt 300 acttcctcat tatactaaat ttaacatcga tgattttattttctgtttct cttctttcca 360 cctacataca tcccaaaatt tagggtgcaa ttttaagtttattaacacat gtttttagct 420 gcatgctgcc tttgtgtgtg ctcaccaaat tgcattcttctctttatatg ttgtatttga 480 attttcacac catatgtaaa caagattacg tacgtgtccatgatcaaata caaatgctgt 540 cttatactgg caatttgata aacagccgtc cattttttctttttctcttt aactatatat 600 gctctagaat ctctgaagat tcctctgcca tcgaatttctttcttggtaa caacgtcgtc 660 gttatgttat tattttattc tatttttatt ttatcatatatatttcttat tttgttcgaa 720 gtatgtcata ttttgatcgt gacaattaga ttgtcatgtaggagtaggaa tatcacttta 780 aaacattgat tagtctgtag gcaatattgt cttctttttcctcctttatt aatatatttt 840 gtcgaagttt taccacaagg ttgattcgct ttttttgtccctttctcttg ttctttttac 900 ctcaggtatt ttagtctttc atggattata agatcactgagaagtgtatg catgtaatac 960 taagcaccat agctgttctg cttgaattta tttgtgtgtaaattgtaatg tttcagcgtt 1020 ggctttccct gtagctgcta caatggtact gtatatctattttttgcatt gttttcattt 1080 tttcttttac ttaatcttca ttgctttgaa attaataaaacaatataata tagtttgaac 1140 tttgaactat tgcctattca tgtaattaac ttattcactgactcttattg tttttctggt 1200 agaattcatt ttaaattgaa ggataaatta agaggcaatacttgtaaatt gacctgtcat 1260 aattacacag gaccctgttt tgtgcctttt tgtctctgtctttggttttg catgttagcc 1320 tcacacagat atttagtagt tgttctgcat acaagcctcacacgtatact aaaccagtgg 1380 acctcaaagt catggcctta cacctattgc atgcgagtctgtgacacaac ccctggtttc 1440 catattgcaa tgtgctacgc cgtcgtcctt gtttgtttccatatgtatat tgataccatc 1500 aaattattat atcatttata tggtctggac cattacgtgtactctttatg acatgtaatt 1560 gagtttttta attaaaaaaa tcaatgaaat ttaactacgtagcatcatat agagataatt 1620 gactagaaat ttgatgactt attctttcct aatcatattttcttgtattg atagccccgc 1680 tgtccctttt aaactcccga gaga 1704 23 4010 DNAGlycine max 23 acaaagcctt tagcctatgc tgccaataat ggataccaac aaaagggttcttcttttgat 60 tttgatccta gcgctcctcc accgtttaag attgcagaaa tcagagcttcaataccaaaa 120 cattgctggg tcaagaatcc atggagatcc ctcagttatg ttctcagggatgtgcttgta 180 attgctgcat tggtggctgc agcaattcac ttcgacaact ggcttctctggctaatctat 240 tgccccattc aaggcacaat gttctgggct ctctttgttc ttggacatgattggtaataa 300 tttttgtgtt tcttactctt tttttttttt ttttgtttat gatatgaatctcacacattg 360 ttctgttatg tcatttcttc ttcatttggc tttagacaac ttaaatttgagatctttatt 420 atgtttttgc ttatatggta aagtgattct tcattatttc attcttcattgattgaattg 480 aacagtggcc atggaagctt ttcagatagc cctttgctga atagcctggtgggacacatc 540 ttgcattcct caattcttgt gccataccat ggatggttag ttcatactggcttttttgtt 600 tgttcatttg tcattgaaaa aaaatctttt gttgattcaa ttatttttatagtgtgtttg 660 gaagcccgtt tgagaaaata agaaatcgca tctggaatgt gaaagttataactatttagc 720 ttcatctgtc gttgcaagtt cttttattgg ttaaattttt atagcgtgctaggaaaccca 780 ttcgagaaaa taagaaatca catctggaat gtgaaagtta taactgttagcttctgagta 840 aacgtggaaa aaccacattt tggatttgga accaaatttt atttgataaatgacaaccaa 900 attgattttg atggattttg caggagaatt agccacagaa ctcaccatgaaaaccatgga 960 cacattgaga aggatgagtc atgggttcca gtatgtgatt aattgcttctcctatagttg 1020 ttcttgattc aattacattt tatttatttg gtaggtccaa gaaaaaagggaatctttatg 1080 cttcctgagg ctgttcttga acatggctct tttttatgtg tcattatcttagttaacaga 1140 gaagatttac aagaatctag acagcatgac aagactcatt agattcactgtgccatttcc 1200 atgtttgtgt atccaattta tttggtgagt gattttttga cttggaagacaacaacacat 1260 tattattata atatggttca aaacaatgac tttttcttta tgatgtgaactccatttttt 1320 agttttcaag aagccccgga aaggaaggct ctcacttcaa tccctacagcaatctgtttc 1380 cacccagtga gagaaaagga atagcaatat caacactgtg ttgggctaccatgttttctc 1440 tgcttatcta tctctcattc attaactagt ccacttctag tgctcaagctctatggaatt 1500 ccatattggg taactaaatt actcctacat tgttactttt tcctccttttttttattatt 1560 tcaattctcc aattggaaat ttgaaatagt taccataatt atgtaattgtttgatcatgt 1620 gcagatgttt gttatgtggc tggactttgt cacatacttg catcaccatggtcaccacca 1680 gaaactgcct tggtaccgcg gcaaggtaac aaaaataaat agaaaatagtgggtgaacac 1740 ttaaatgcga gatagtaata cctaaaaaaa gaaaaaaata taggtataataaataatata 1800 actttcaaaa taaaaagaaa tcatagagtc tagcgtagtg tttggagtgaaatgatgttc 1860 acctaccatt actcaaagat tttgttgtgt cccttagttc attcttattattttacatat 1920 cttacttgaa aagacttttt aattattcat tgagatctta aagtgactgttaaattaaaa 1980 taaaaaacaa gtttgttaaa acttcaaata aataagagtg aagggagtgtcatttgtctt 2040 ctttctttta ttgcgttatt aatcacgttt ctcttctctt ttttttttttcttctctgct 2100 ttccacccat tatcaagttc atgtgaagca gtggcggatc tatgtaaatgagtggggggc 2160 aattgcaccc acaagatttt attttttatt tgtacaggaa taataaaataaaactttgcc 2220 cccataaaaa ataaatattt tttcttaaaa taatgcaaaa taaatataagaaataaaaag 2280 agaataaatt attattaatt ttattatttt gtacttttta tttagtttttttagcggtta 2340 gatttttttt tcatgacatt atgtaatctt ttaaaagcat gtaatatttttattttgtga 2400 aaataaatat aaatgatcat attagtctca gaatgtataa actaataataattttatcac 2460 taaaagaaat tctaatttag tccataaata agtaaaacaa gtgacaattatattttatat 2520 ttacttaatg tgaaataata cttgaacatt ataataaaac ttaatgacaggagatattac 2580 atagtgccat aaagatattt taaaaaataa aatcattaat acactgtactactatataat 2640 attcgatata tatttttaac atgattctca atagaaaaat tgtattgattatattttatt 2700 agacatgaat ttacaagccc cgtttttcat ttatagctct tacctgtgatctattgtttt 2760 gcttcgctgt ttttgttggt caagggactt agatgtcaca atattaatactagaagtaaa 2820 tatttatgaa aacatgtacc ttacctcaac aaagaaagtg tggtaagtggcaacacacgt 2880 gttgcatttt tggcccagca ataacacgtg tttttgtggt gtactaaaatggacaggaat 2940 ggagttattt aagaggtggc ctcaccactg tggatcgtga ctatggttggatcaataaca 3000 ttcaccatga cattggcacc catgttatcc accatctttt cccccaaattcctcattatc 3060 acctcgttga agcggtacat tttattgctt attcacctaa aaacaatacaattagtacat 3120 ttgttttatc tcttggaagt tagtcatttt cagttgcatg attctaatgctctctccatt 3180 cttaaatcat gttttcacac ccacttcatt taaaataaga acgtgggtgttattttaatt 3240 tctattcact aacatgagaa attaacttat ttcaagtaat aattttaaaatatttttatg 3300 ctattatttt attacaaata attatgtata ttaagtttat tgattttataataattatat 3360 taaaattata tcgatattaa tttttgattc actgatagtg ttttatattgttagtactgt 3420 gcatttattt taaaattggc ataaataata tatgtaacca gctcactatactatactggg 3480 agcttggtgg tgaaaggggt tcccaaccct cctttctagg tgtacatgctttgatacttc 3540 tggtaccttc ttatatcaat ataaattata ttttgctgat aaaaaaacatggttaaccat 3600 taaattcttt ttttaaaaaa aaaactgtat ctaaactttg tattattaaaaagaagtctg 3660 agattaacaa taaactaaca ctcatttgga ttcactgcag acacaagcagcaaaaccagt 3720 tcttggagat tactaccgtg agccagaaag atctgcgcca ttaccatttcatctaataaa 3780 gtatttaatt cagagtatga gacaagacca cttcgtaagt gacactggagatgttgttta 3840 ttatcagact gattctctgc tcctccactc gcaacgagac tgagtttcaaactttttggg 3900 ttattattta ttgattctag ctactcaaat tacttttttt ttaatgttatgttttttgga 3960 gtttaacgtt ttctgaacaa cttgcaaatt acttgcatag agagacatgg4010 24 34 DNA Artificial sequence PCR primer 24 acgaattcct cgaggtaaattaaattgtgc ctgc 34 25 33 DNA Artificial sequence PCR primer 25gcgagatcta tcgatctgtg tcaaagtata aac 33 26 19 DNA Artificial sequencePCR primer 26 catgctttct gtgcttctc 19 27 19 DNA Artificial sequence PCRprimer 27 gttgatccaa ccatagtcg 19 28 36 DNA Artificial sequence PCRprimer 28 gcgatcgatg tatgatgcta aattaaattg tgcctg 36 29 30 DNAArtificial sequence PCR primer 29 gcggaattcc tgtgtcaaag tataaagaag 30 3030 DNA Artificial sequence PCR primer 30 gatcgatgcc cggggtaataatttttgtgt 30 31 29 DNA Artificial sequence PCR primer 31 cacgcctcgagtgttcaatt caatcaatg 29 32 24 DNA Artificial sequence PCR primer 32cactcgagtt agttcatact ggct 24 33 25 DNA Artificial sequence PCR primer33 cgcatcgatt gcaaaatcca tcaaa 25 34 38 DNA Artificial sequence PCRprimer 34 cuacuacuac uactcgagcg taaatagtgg gtgaacac 38 35 41 DNAArtificial sequence PCR primer 35 caucaucauc auctcgagga attcgtccattttagtacac c 41 36 39 DNA Artificial sequence PCR primer 36 cuacuacuacuactcgaggc gcgtacattt tattgctta 39 37 41 DNA Artificial sequence PCRprimer 37 caucaucauc auctcgagga attctgcagt gaatccaaat g 41 38 22 DNAArtificial sequence PCR primer 38 caccatggtc atcatcagaa ac 22 39 22 DNAArtificial sequence PCR primer 39 tcacgatcca cagttgtgag ac 22 40 4086DNA Glycine max soybean FATB genomic clone 40 ttagggaaac aacaaggacgcaaaatgaca caatagccct tcttccctgt ttccagcttt 60 tctccttctc tctctccatcttcttcttct tcttcactca gtcaggtacg caaacaaatc 120 tgctattcat tcattcattcctctttctct ctgatcgcaa actgcacctc tacgctccac 180 tcttctcatt ttctcttcctttctcgcttc tcagatccaa ctcctcagat aacacaagac 240 caaacccgct ttttctgcatttctagacta gacgttctac cggagaaggt tctcgattct 300 tttctctttt aactttatttttaaaataat aataatgaga gctggatgcg tctgttcgtt 360 gtgaatttcg aggcaatggggttctcattt tcgttacagt tacagattgc attgtctgct 420 ttcctcttct cccttgtttctttgccttgt ctgatttttc gtttttattt cttactttta 480 atttttgggg atggatattttttctgcatt ttttcggttt gcgatgtttt caggattccg 540 attccgagtc agatctgcgccggcttatac gacgaatttg ttcttattcg caacttttcg 600 cttgattggc ttgttttacctctggaatct cacacgtgat caaataagcc tgctatttta 660 gttgaagtag aatttgttctttatcggaaa gaattctatg gatctgttct gaaattggag 720 ctactgtttc gagttgctattttttttagt agtattaaga acaagtttgc cttttatttt 780 acattttttt cctttgcttttgccaaaagt ttttatgatc actctcttct gtttgtgata 840 taactgatgt gctgtgctgttattatttgt tatttggggt gaagtataat tttttgggtg 900 aacttggagc atttttagtccgattgattt ctcgatatca tttaaggcta aggttgacct 960 ctaccacgcg tttgcgtttgatgttttttc catttttttt ttatctcata tcttttacag 1020 tgtttgccta tttgcatttctcttctttat cccctttctg tggaaaggtg ggagggaaaa 1080 tgtatttttt ttttctcttctaacttgcgt atattttgca tgcagcgacc ttagaaattc 1140 attatggtgg caacagctgctacttcatca tttttccctg ttacttcacc ctcgccggac 1200 tctggtggag caggcagcaaacttggtggt gggcctgcaa accttggagg actaaaatcc 1260 aaatctgcgt cttctggtggcttgaaggca aaggcgcaag ccccttcgaa aattaatgga 1320 accacagttg ttacatctaaagaaggcttc aagcatgatg atgatctacc ttcgcctccc 1380 cccagaactt ttatcaaccagttgcctgat tggagcatgc ttcttgctgc tatcacaaca 1440 attttcttgg ccgctgaaaagcagtggatg atgcttgatt ggaagccacg gcgacctgac 1500 atgcttattg acccctttgggataggaaaa attgttcagg atggtcttgt gttccgtgaa 1560 aacttttcta ttagatcatatgagattggt gctgatcgta ccgcatctat agaaacagta 1620 atgaaccatt tgcaagtaagtccgtcctca tacaagtgaa tctttatgat cttcagagat 1680 gagtatgctt tgactaagatagggctgttt atttagacac tgtaattcaa tttcatatat 1740 agataatatc attctgttgttacttttcat actatattta tatcaactat ttgcttaaca 1800 acaggaaact gcacttaatcatgttaaaag tgctgggctt cttggtgatg gctttggttc 1860 cacgccagaa atgtgcaaaaagaacttgat atgggtggtt actcggatgc aggttgtggt 1920 ggaacgctat cctacatggttagtcatcta gattcaacca ttacatgtga tttgcaatgt 1980 atccatgtta agctgctatttctctgtcta ttttagtaat ctttatgagg aatgatcact 2040 cctaaatata ttcatggtaattattgagac ttaattatga gaaccaaaat gctttggaaa 2100 tttgtctggg atgaaaattgattagataca caagctttat acatgatgaa ctatgggaaa 2160 ccttgtgcaa cagagctattgatctgtaca agagatgtag tatagcatta attacatgtt 2220 attagataag gtgacttatccttgtttaat tattgtaaaa atagaagctg atactatgta 2280 ttctttgcat ttgttttcttaccagttata tataccctct gttctgtttg agtactacta 2340 gatgtataaa gaatgcaattattctgactt cttggtgttg ggttgaagtt agataagcta 2400 ttagtattat tatggttattctaaatctaa ttatctgaaa ttgtgtgtct atatttgctt 2460 caggggtgac atagttcaagtggacacttg ggtttctgga tcagggaaga atggtatgcg 2520 tcgtgattgg cttttacgtgactgcaaaac tggtgaaatc ttgacaagag cttccaggta 2580 gaaatcattc tctgtaattttccttcccct ttccttctgc ttcaagcaaa ttttaagatg 2640 tgtatcttaa tgtgcacgatgctgattgga cacaatttta aatctttcaa acatttacaa 2700 aagttatgga accctttcttttctctcttg aagatgcaaa tttgtcacga ctgaagtttg 2760 aggaaatcat ttgaattttgcaatgttaaa aaagataatg aactacatat tttgcaggca 2820 aaaacctcta attgaacaaactgaacattg tatcttagtt tatttatcag actttatcat 2880 gtgtactgat gcatcaccttggagcttgta atgaattaca tattagcatt ttctgaactg 2940 tatgttatgg ttttggtgatctacagtgtt tgggtcatga tgaataagct gacacggagg 3000 ctgtctaaaa ttccagaagaagtcagacag gagataggat cttattttgt ggattctgat 3060 ccaattctag aagaggataacagaaaactg actaaacttg acgacaacac agcggattat 3120 attcgtaccg gtttaagtgtatgtcaacta gtttttttgt aattgttgtc attaatttct 3180 tttcttaaat tatttcagatgttgctttct aattagttta cattatgtat cttcattctt 3240 ccagtctagg tggagtgatctagatatcaa tcagcatgtc aacaatgtga agtacattga 3300 ctggattctg gaggtatttttctgttcttg tattctaatc cactgcagtc cttgttttgt 3360 tgttaaccaa aggactgtcctttgattgtt tgcagagtgc tccacagcca atcttggaga 3420 gtcatgagct ttcttccgtgactttagagt ataggaggga gtgtggtagg gacagtgtgc 3480 tggattccct gactgctgtatctggggccg acatgggcaa tctagctcac agtggacatg 3540 ttgagtgcaa gcatttgcttcgactcgaaa atggtgctga gattgtgagg ggcaggactg 3600 agtggaggcc caaacctatgaacaacattg gtgttgtgaa ccaggttcca gcagaaagca 3660 cctaagattt tgaaatggttaacggttgga gttgcatcag tctccttgct atgtttagac 3720 ttattctggc ctctggggagagttttgctt gtgtctgtcc aatcaatcta catatcttta 3780 tatccttcta atttgtgttactttggtggg taagggggaa aagctgcagt aaacctcatt 3840 ctctctttct gctgctccatatttcatttc atctctgatt gcgctactgc taggctgtct 3900 tcaatattta attgcttgatcaaaatagct aggcatgtat attattattc ttttctcttg 3960 gctcaattaa agatgcaattttcattgtga acacagcata actattattc ttattatttt 4020 tgtatagcct gtatgcacgaatgacttgtc catccaatac aaccgtgatt gtatgctcca 4080 gctcag 4086 41 109 DNAGlycine max FATB intron I 41 gtacgcaaac aaatctgcta ttcattcatt cattcctctttctctctgat cgcaaactgc 60 acctctacgc tccactcttc tcattttctc ttcctttctcgcttctcag 109 42 836 DNA Glycine max FATB intron II 42 gttctcgattcttttctctt ttaactttat ttttaaaata ataataatga gagctggatg 60 cgtctgttcgttgtgaattt cgaggcaatg gggttctcat tttcgttaca gttacagatt 120 gcattgtctgctttcctctt ctcccttgtt tctttgcctt gtctgatttt tcgtttttat 180 ttcttacttttaatttttgg ggatggatat tttttctgca ttttttcggt ttgcgatgtt 240 ttcaggattccgattccgag tcagatctgc gccggcttat acgacgaatt tgttcttatt 300 cgcaacttttcgcttgattg gcttgtttta cctctggaat ctcacacgtg atcaaataag 360 cctgctattttagttgaagt agaatttgtt ctttatcgga aagaattcta tggatctgtt 420 ctgaaattggagctactgtt tcgagttgct atttttttta gtagtattaa gaacaagttt 480 gccttttattttacattttt ttcctttgct tttgccaaaa gtttttatga tcactctctt 540 ctgtttgtgatataactgat gtgctgtgct gttattattt gttatttggg gtgaagtata 600 attttttgggtgaacttgga gcatttttag tccgattgat ttctcgatat catttaaggc 660 taaggttgacctctaccacg cgtttgcgtt tgatgttttt tccatttttt ttttatctca 720 tatcttttacagtgtttgcc tatttgcatt tctcttcttt atcccctttc tgtggaaggt 780 gggagggaaaatgtattttt tttttctctt ctaacttgcg tatattttgc atgcag 836 43 169 DNAGlycine max FATB intron III 43 gtaagtccgt cctcatacaa gtgaatctttatgatcttca gagatgagta tgctttgact 60 aagatagggc tgtttattta gacactgtaattcaatttca tatatagata atatcattct 120 gttgttactt ttcatactat atttatatcaactatttgct taacaacag 169 44 328 PRT Glycine max soybean FATB enzyme 44Met Glu Glu Gln Leu Leu Ala Ala Ile Thr Thr Ile Phe Leu Ala Ala 1 5 1015 Glu Lys Gln Trp Met Met Leu Asp Trp Lys Pro Arg Arg Pro Asp Met 20 2530 Leu Ile Asp Pro Phe Gly Ile Gly Lys Ile Val Gln Asp Gly Leu Val 35 4045 Phe Arg Glu Asn Phe Ser Ile Arg Ser Tyr Glu Ile Gly Ala Asp Arg 50 5560 Thr Ala Ser Ile Glu Thr Val Met Asn His Leu Gln Glu Thr Ala Leu 65 7075 80 Asn His Val Lys Ser Ala Gly Leu Leu Gly Asp Gly Phe Gly Ser Thr 8590 95 Pro Glu Met Cys Lys Lys Asn Leu Ile Trp Val Val Thr Arg Met Gln100 105 110 Val Val Val Glu Arg Tyr Pro Thr Trp Gly Asp Ile Val Gln ValAsp 115 120 125 Thr Trp Val Ser Gly Ser Gly Lys Asn Gly Met Arg Arg AspTrp Leu 130 135 140 Leu Arg Asp Ser Lys Thr Gly Glu Ile Leu Thr Arg AlaSer Ser Val 145 150 155 160 Trp Val Met Met Asn Lys Leu Thr Arg Arg LeuSer Lys Ile Pro Glu 165 170 175 Glu Val Arg Gln Glu Ile Gly Ser Tyr PheVal Asp Ser Asp Pro Ile 180 185 190 Leu Glu Glu Asp Asn Arg Lys Leu ThrLys Leu Asp Asp Asn Thr Ala 195 200 205 Asp Tyr Ile Arg Thr Gly Leu SerPro Arg Trp Ser Asp Leu Asp Ile 210 215 220 Asn Gln His Val Asn Asn ValLys Tyr Ile Gly Trp Ile Leu Glu Ser 225 230 235 240 Ala Pro Gln Pro IleLeu Glu Ser His Glu Leu Ser Ser Met Thr Leu 245 250 255 Glu Tyr Arg ArgGlu Cys Gly Arg Asp Ser Val Leu Asp Ser Leu Thr 260 265 270 Ala Val SerGly Ala Asp Met Gly Asn Leu Ala His Ser Gly His Val 275 280 285 Glu CysLys His Leu Leu Arg Leu Glu Asn Gly Ala Glu Ile Val Arg 290 295 300 GlyArg Thr Glu Trp Arg Pro Lys Pro Val Asn Asn Phe Gly Val Val 305 310 315320 Asn Gln Val Pro Ala Glu Ser Thr 325 45 1856 DNA Glycine max soybeanFATB partial genomic clone 45 ttagggaaac aacaaggacg caaaatgacacaatagccct tcttccctgt ttccagcttt 60 tctccttctc tctctccatc ttcttcttcttcttcactca gtcaggtacg caaacaaatc 120 tgctattcat tcattcattc ctctttctctctgatcgcaa actgcacctc tacgctccac 180 tcttctcatt ttctcttcct ttctcgcttctcagatccaa ctcctcagat aacacaagac 240 caaacccgct ttttctgcat ttctagactagacgttctac cggagaaggt tctcgattct 300 tttctctttt aactttattt ttaaaataataataatgaga gctggatgcg tctgttcgtt 360 gtgaatttcg aggcaatggg gttctcattttcgttacagt tacagattgc attgtctgct 420 ttcctcttct cccttgtttc tttgccttgtctgatttttc gtttttattt cttactttta 480 atttttgggg atggatattt tttctgcattttttcggttt gcgatgtttt caggattccg 540 attccgagtc agatctgcgc cggcttatacgacgaatttg ttcttattcg caacttttcg 600 cttgattggc ttgttttacc tctggaatctcacacgtgat caaataagcc tgctatttta 660 gttgaagtag aatttgttct ttatcggaaagaattctatg gatctgttct gaaattggag 720 ctactgtttc gagttgctat tttttttagtagtattaaga acaagtttgc cttttatttt 780 acattttttt cctttgcttt tgccaaaagtttttatgatc actctcttct gtttgtgata 840 taactgatgt gctgtgctgt tattatttgttatttggggt gaagtataat tttttgggtg 900 aacttggagc atttttagtc cgattgatttctcgatatca tttaaggcta aggttgacct 960 ctaccacgcg tttgcgtttg atgttttttccatttttttt ttatctcata tcttttacag 1020 tgtttgccta tttgcatttc tcttctttatcccctttctg tggaaggtgg gagggaaaat 1080 gtattttttt tttctcttct aacttgcgtatattttgcat gcagcgacct tagaaattca 1140 ttatggtggc aacagctgct acttcatcatttttccctgt tacttcaccc tcgccggact 1200 ctggtggagc aggcagcaaa cttggtggtgggcctgcaaa ccttggagga ctaaaatcca 1260 aatctgcgtc ttctggtggc ttgaaggcaaaggcgcaagc cccttcgaaa attaatggaa 1320 ccacagttgt tacatctaaa gaaggcttcaagcatgatga tgatctacct tcgcctcccc 1380 ccagaacttt tatcaaccag ttgcctgattggagcatgct tcttgctgct atcacaacaa 1440 ttttcttggc cgctgaaaag cagtggatgatgcttgattg gaagccacgg cgacctgaca 1500 tgcttattga cccctttggg ataggaaaaattgttcagga tggtcttgtg ttccgtgaaa 1560 acttttctat tagatcatat gagattggtgctgatcgtac cgcatctata gaaacagtaa 1620 tgaaccattt gcaagtaagt ccgtcctcatacaagtgaat ctttatgatc ttcagagatg 1680 agtatgcttt gactaagata gggctgtttatttagacact gtaattcaat ttcatatata 1740 gataatatca ttctgttgtt acttttcatactatatttat atcaactatt tgcttaacaa 1800 caggaaactg cacttaatca tgttaaaagtgctgggcttc ttggtgatgg ctggta 1856 46 34 DNA Artificial Oligonucleotideprimer F1 46 gcggccgccc cgggttaggg aaacaacaag gacg 34 47 34 DNAArtificial Oligonucleotide primer F2 47 gcggccgccc cgggcagtca gatccaactcctca 34 48 34 DNA Artificial Oligonucleotide primer F3 48 gcggccgccccgggattggt gctgatcgta ccgc 34 49 38 DNA Artificial Oligonucleotideprimer R1 49 gcggccgcgg taccccccct tacccaccaa agtatcac 38 50 34 DNAArtificial Oligonucleotide primer R2 50 gcggccgcgg taccaaactc tccccagggaacca 34 51 34 DNA Artificial Oligonucleotide primer R3 51 gcggccgcggtaccagccat caccaagaag ccca 34 52 37 DNA Artificial Oligonucleotideprimer 18133 52 gaattcctcg agctcgattc ttttctcttt taacttt 37 53 37 DNAArtificial Oligonucleotide primer 18134 53 gaattcctcg agcatgcaaaatatacgcaa gttagaa 37 54 854 DNA Artificial PCR product containingsoybean FATB intron II 54 gaattcctcg agctcgattc ttttctcttt taactttatttttaaaataa taataatgag 60 agctggatgc gtctgttcgt tgtgaatttc gaggcaatggggttctcatt ttcgttacag 120 ttacagattg cattgtctgc tttcctcttc tcccttgtttctttgccttg tctgattttt 180 cgtttttatt tcttactttt aatttttggg gatggatattttttctgcat tttttcggtt 240 tgcgatgttt tcaggattcc gattccgagt cagatctgcgccggcttata cgacgaattt 300 gttcttattc gcaacttttc gcttgattgg cttgttttacctctggaatc tcacacgtga 360 tcaaataagc ctgctatttt agttgaagta gaatttgttctttatcggaa agaattctat 420 ggatctgttc tgaaattgga gctactgttt cgagttgctattttttttag tagtattaag 480 aacaagtttg ccttttattt tacatttttt tcctttgcttttgccaaaag tttttatgat 540 cactctcttc tgtttgtgat ataactgatg tgctgtgctgttattatttg ttatttgggg 600 tgaagtataa ttttttgggt gaacttggag catttttagtccgattgatt tctcgatatc 660 atttaaggct aaggttgacc tctaccacgc gtttgcgtttgatgtttttt ccattttttt 720 tttatctcat atcttttaca gtgtttgcct atttgcatttctcttcttta tcccctttct 780 gtggaaggtg ggagggaaaa tgtatttttt ttttctcttctaacttgcgt atattttgca 840 tgctcgagga attc 854 55 1688 DNA Glycine maxsoybean FATB cDNA 55 acaattacac tgtctctctc ttttccaaaa ttagggaaacaacaaggacg caaaatgaca 60 caatagccct tcttccctgt ttccagcttt tctccttctctctctctcca tcttcttctt 120 cttcttcact cagtcagatc caactcctca gataacacaagaccaaaccc gctttttctg 180 catttctaga ctagacgttc taccggagaa gcgaccttagaaattcatta tggtggcaac 240 agctgctact tcatcatttt tccctgttac ttcaccctcgccggactctg gtggagcagg 300 cagcaaactt ggtggtgggc ctgcaaacct tggaggactaaaatccaaat ctgcgtcttc 360 tggtggcttg aaggcaaagg cgcaagcccc ttcgaaaattaatggaacca cagttgttac 420 atctaaagaa agcttcaagc atgatgatga tctaccttcgcctcccccca gaacttttat 480 caaccagttg cctgattgga gcatgcttct tgctgctatcacaacaattt tcttggccgc 540 tgaaaagcag tggatgatgc ttgattggaa gccacggcgacctgacatgc ttattgaccc 600 ctttgggata ggaaaaattg ttcaggatgg tcttgtgttccgtgaaaact tttctattag 660 atcatatgag attggtgctg atcgtaccgc atctatagaaacagtaatga accatttgca 720 agaaactgca cttaatcatg ttaaaagtgc tgggcttcttggtgatggct ttggttccac 780 gccagaaatg tgcaaaaaga acttgatatg ggtggttactcggatgcagg ttgtggtgga 840 acgctatcct acatggggtg acatagttca agtggacacttgggtttctg gatcagggaa 900 gaatggtatg cgtcgtgatt ggcttttacg tgactccaaaactggtgaaa tcttgacaag 960 agcttccagt gtttgggtca tgatgaataa gctaacacggaggctgtcta aaattccaga 1020 agaagtcaga caggagatag gatcttattt tgtggattctgatccaattc tggaagagga 1080 taacagaaaa ctgactaaac ttgacgacaa cacagcggattatattcgta ccggtttaag 1140 tcctaggtgg agtgatctag atatcaatca gcatgtcaacaatgtgaagt acattggctg 1200 gattctggag agtgctccac agccaatctt ggagagtcatgagctttctt ccatgacttt 1260 agagtatagg agagagtgtg gtagggacag tgtgctggattccctgactg ctgtatctgg 1320 ggccgacatg ggcaatctag ctcacagcgg gcatgttgagtgcaagcatt tgcttcgact 1380 ggaaaatggt gctgagattg tgaggggcag gactgagtggaggcccaaac ctgtgaacaa 1440 ctttggtgtt gtgaaccagg ttccagcaga aagcacctaagatttgaaat ggttaacgat 1500 tggagttgca tcagtctcct tgctatgttt agacttattctggttccctg gggagagttt 1560 tgcttgtgtc tatccaatca atctacatgt ctttaaatatatacaccttc taatttgtga 1620 tactttggtg ggtaaggggg aaaagcagca gtaaatctcattctcattgt aattaaaaaa 1680 aaaaaaaa 1688

What is claimed is:
 1. A nucleic acid construct comprising DNA which istranscribed into RNA that forms at least one double-stranded RNAmolecule, wherein one strand of said double-stranded molecule is codedby a portion of said DNA which is at least 90% identical to at least onetranscribed intron of a gene.
 2. The construct of claim 1, wherein onestrand of said double-stranded molecule is coded by a portion of saidDNA which is at least 98% identical to at least one transcribed intronof a gene.
 3. The construct of claim 1, wherein one strand of saiddouble-stranded molecule is coded by a portion of said DNA which is 100%identical to at least one transcribed intron of a gene.
 4. The constructof claim 1, comprising in series one strand of an intron, a spliceableintron, and the complement of said intron, wherein said spliceableintron provides a hairpin structure, and wherein said intron and saidcomplement of said intron can hybridize to each other.
 5. The constructof claim 1, wherein said transcribed introns are in FAD2 genes or FAD3genes.
 6. The construct of claim 1, comprising DNA which is transcribedinto RNA that forms at least one double-stranded RNA molecule whereinone strand of said double-stranded molecule is coded by a portion ofsaid DNA which is at least 90% identical to at least two transcribedintrons.
 7. The construct of claim 6, comprising DNA which istranscribed into RNA that forms two or more double-stranded RNAmolecules.
 8. A transformed cell or organism having in its genome anintroduced nucleic acid construct comprising DNA which is transcribedinto RNA that forms at least one double-stranded RNA molecule, whereinone strand of said double-stranded molecule is coded by a portion ofsaid DNA which is at least 90% identical to at least one transcribedintron of a gene.
 9. A transformed plant having in its genome anintroduced nucleic acid construct comprising DNA which is transcribedinto RNA that forms at least one double-stranded RNA molecule, whereinone strand of said double-stranded molecule is coded by a portion ofsaid DNA which is at least 90% identical to at least one transcribedintron of a gene.
 10. The transformed plant of claim 9, having in itsgenome an introduced nucleic acid construct comprising DNA which istranscribed into RNA that forms at least one double-stranded RNAmolecule wherein one strand of said double-stranded molecule is coded bya portion of said DNA which is at least 98% identical to at least onetranscribed intron of a native plant gene.
 11. The transformed plant ofclaim 9, wherein said intron is from a FAD2 gene or a FAD3 gene.
 12. Thetransformed plant of claim 11, wherein expression of a protein encodedby said FAD2 gene or said FAD3 gene is reduced.
 13. The transformedplant of claim 11, wherein expression of a protein encoded by said FAD2gene or said FAD3 gene is substantially reduced.
 14. The transformedplant of claim 11, wherein expression of the protein encoded by saidFAD2 gene or said FAD3 gene is effectively eliminated.
 15. A method ofreducing expression of a protein encoded by a target gene in a mammalcomprising introducing into a cell or organism a nucleic acid constructcomprising DNA which is transcribed into RNA that forms at least onedouble-stranded RNA molecule, wherein one strand of said double-strandedmolecule is coded by a portion of said DNA which is at least 90%identical to at least one transcribed intron of a gene.
 16. The methodof claim 15, wherein the target gene encodes a protein in an insect ornematode which is a pest to a plant, and wherein said method comprisesintroducing into the genome of said plant a nucleic acid constructcomprising DNA which is transcribed into RNA that forms at least onedouble-stranded RNA molecule which is effective for reducing expressionof said target gene when said insect or nematode ingests cells from saidplant.
 17. A method of reducing expression of a protein encoded by atarget gene in a plant comprising introducing into a plant genome anucleic acid construct comprising DNA which is transcribed into RNA thatforms at least one double-stranded RNA molecule, wherein one strand ofsaid double-stranded molecule is coded by a portion of said DNA which isat least 90% identical to at least one transcribed intron of a gene.